Inhibitors of mutant isocitrate dehydrogenases and compositions and methods thereof

ABSTRACT

The invention provides novel chemical compounds useful for treating cancer, or a related disease or disorder thereof, and pharmaceutical composition and methods of preparation and use thereof.

PRIORITY CLAIMS AND RELATED PATENT APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication Ser. Nos. 62/406,622, filed on Oct. 11, 2016, and62/466,453, filed Mar. 3, 2017, the entire content of each of which isincorporated herein by reference in its entirety.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to therapeutics and treatment methodsfor certain diseases and conditions. More particularly, the inventionprovides novel chemical compounds and pharmaceutical compositionsthereof useful for treating cancer and methods of preparation and usethereof.

BACKGROUND OF THE INVENTION

Isocitrate dehydrogenase (IDH) is an enzyme that catalyzes the oxidativedecarboxylation of isocitrate, producing alpha-ketoglutarate(α-ketoglutarate) and CO₂. IDH exists in three isoforms in humans: IDH3catalyzes the third step of the citric acid cycle while converting NAD+to NADH in the mitochondria. The isoforms IDH1 and IDH2 catalyze thesame reaction outside the context of the citric acid cycle and use NADP+as a cofactor instead of NAD+. IDHs localize to the cytosol as well asthe mitochondrion and peroxisome.

Normal, wild type IDH enzymes help to break down nutrients and generateenergy for cells. When mutated, IDH creates a molecule that alters thecells' genetic programming, and instead of maturing, the cells remainprimitive and proliferate quickly. Non-mutant IDH 1/2 catalyzes theoxidative decarboxylation of isocitrate to a-ketoglutarate (a-KG)thereby reducing NAD+ (NADP+) to NADP (NADPH), e.g., in the forwardreaction.

IDH1 and IDH2 are mutated in a wide range of hematologic and solid tumormalignancies. Mutations of IDH 1/2 present in certain cancer cellsresult in a new ability of the enzyme to catalyze the NAPH-dependentreduction of -ketoglutarate to R (-)-2-hydroxyglutarate (2HG), which isnot formed by wild-type IDH 1/2. Human IDH2 gene encodes a protein of452 amino acids. (GenBank entries NM_002168.2 and NP_002159.2; The MGCProject Team 2004, Genome Res. 14:2121-2127). Human IDH1 gene encodes aprotein of 414 amino acids (GenBank entries NM_005896.2 and NP_005887.2;Nekrutenko et al, 1998 Mol. Biol. Evol. 15:1674-1684; Geisbrecht et al,1999 J. Biol. Chem. 274:30527-30533; Wiemann et al, 2001 Genome Res.11:422-435; The MGC Project Team 2004 Genome Res. 14:2121-2127; Sjoeblomet al. 2006 Science 314:268-274.) 2HG production is believed tocontribute to the formation and progression of cancer. (Dang, et al.2009 Nature 462:739-44.)

There is an urgent and growing need for improved cancer therapeutics andtreatment methods, e.g., via effective inhibition of mutant IDH 1/2 andtheir alpha hydroxyl neoactivity.

SUMMARY OF THE INVENTION

The invention provides novel, orally available, selective and potentinhibitors of mutated IDH 1 and/or IDH 2 proteins. The compoundsdisclosed herein form irreversible covalent bond or reversible bindinginteractions with mutant IDH 1 and/or IDH 2 protein and effectivelyinhibit their respective alpha hydroxyl neoactivity.

In one aspect, the invention generally relates to a compound having thestructural formula of (I):

wherein,

-   -   each of Ring A, Ring B and Ring C is independently a 5- or        6-membered cyclic or bicyclic alkyl, heteroalkyl, aryl, or        heteroaryl ring;    -   each R_(W) is independently a hydrogen, a halide, OH, NH₂,        alkyl, alkyl amine, alkyl amide, alkoxy, or an electrophilic        group R_(E);    -   R₁ is H or a C₁-C₃ alkyl group;    -   X is CH₂, NH, O, S or a single bond;    -   Y is CH₂, NH, O or S; and    -   Z is CH₂, NH, O or S.

In another aspect, the invention generally relates to a pharmaceuticalcomposition comprising a compound having the structural formula of (I):

wherein,

-   -   each of Ring A, Ring B and Ring C is independently a 5- or        6-membered cyclic or bicyclic alkyl, heteroalkyl, aryl, or        heteroaryl ring;    -   each R_(W) is independently a hydrogen, a halide, OH, NH₂,        alkyl, alkyl amine, alkyl amide, alkoxy, or an electrophilic        group R_(E);    -   R₁ is H or a C₁-C₃ alkyl group;    -   X is CH₂, NH, O, S or a single bond;    -   Y is CH₂, NH, O or S; and    -   Z is CH₂, NH, O or S,        or a pharmaceutically acceptable form thereof, effective to        treat, prevent, or reduce one or more cancers, or a related        disease or disorder thereof, in a mammal, including a human, and        a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to apharmaceutical composition comprising a compound disclosed herein.

In yet another aspect, the invention generally relates to a unit dosageform comprising a pharmaceutical composition disclosed herein.

In yet another aspect, the invention generally relates to a method fortreating, reducing, or preventing a disease or disorder, comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising a compound having the structural formula of (I):

wherein,

-   -   each of Ring A, Ring B and Ring C is independently a 5- or        6-membered cyclic or bicyclic alkyl, heteroalkyl, aryl, or        heteroaryl ring;    -   each R_(W) is independently a hydrogen, a halide, OH, NH₂,        alkyl, alkyl amine, alkyl amide, alkoxy, or an electrophilic        group R_(E);    -   R₁ is H or a C₁-C₃ alkyl group;    -   X is CH₂, NH, O, S or a single bond;    -   Y is CH₂, NH, O or S; and    -   Z is CH₂, NH, O or S,        or a pharmaceutically acceptable form thereof, effective to        treat, prevent, or reduce one or more cancers, or a related        disease or disorder thereof, in a mammal, including a human, and        a pharmaceutically acceptable excipient, carrier, or diluent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Crystal structure of IDH1 with GSK321 from PDB:5DE1. FIG. 1A,the cysteine 379 is showed as a targetable residue; FIG. 1B the cysteine269 is showed as targetable residue

FIG. 2. Exemplary IC₅₀ data of representative compound in IDH1 R132Hmutant biochemical assays.

FIG. 3. Exemplary IC₅₀ data of representative compound in IDH1 R132Hmutant biochemical assays.

FIG. 4. Exemplary IC₅₀ data of representative compound in IDH1 R132Cmutant biochemical assays.

FIG. 5. Exemplary IC₅₀ data of representative compound in IDH1 R132Cmutant biochemical assays.

FIG. 6. Exemplary IC₅₀ data of representative compound in IDH1 WTbiochemical assays.

FIG. 7. Exemplary IC₅₀ data of representative compound in IDH1 WTbiochemical assays.

FIG. 8. Exemplary IDH1 R132C intact mass.

FIG. 9. Exemplary intact mass spectrum of IDH1R132C with ISO-2-30treatment.

FIG. 10. Exemplary intact mass spectrum of IDH1R132C with ISO-2-38(another name in mass spectrum:ISO-3-33).

FIG. 11. Exemplary trypsin digestion of labeled IDH1 R132C protein withISO-2-38 (another name in mass spectrum:ISO-3-33).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. General principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in “Organic Chemistry”, Thomas Sorrell, University ScienceBooks, Sausalito: 2006.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios arecontemplated by the present invention. Those of ordinary skill in theart will readily appreciate that analogous ratios are contemplated formore complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic methods well known in the art, and subsequent recoveryof the pure enantiomers.

As used herein, “administration” of a disclosed compound encompasses thedelivery to a subject of a compound as described herein, or a prodrug orother pharmaceutically acceptable derivative thereof, using any suitableformulation or route of administration, as discussed herein.

As used herein, the term “electrophilic group” or “electrophile” refersto group or moiety that is attracted towards and capable of accepting apair of electrons to form a new covalent bond. Exemplary electrophilicgroups include an acrylamide group.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” refer to that amount of a compound or pharmaceuticalcomposition described herein that is sufficient to effect the intendedapplication including, but not limited to, disease treatment, asillustrated below. In some embodiments, the amount is that effective fordetectable killing or inhibition of the growth or spread of cancercells; the size or number of tumors; or other measure of the level,stage, progression or severity of the cancer. The therapeuticallyeffective amount can vary depending upon the intended application, orthe subject and disease condition being treated, e.g., the desiredbiological endpoint, the pharmacokinetics of the compound, the diseasebeing treated, the mode of administration, and the weight and age of thepatient, which can readily be determined by one of ordinary skill in theart. The term also applies to a dose that will induce a particularresponse in target cells, e.g., reduction of cell migration. Thespecific dose will vary depending on, for example, the particularcompounds chosen, the species of subject and their age/existing healthconditions or risk for health conditions, the dosing regimen to befollowed, the severity of the disease, whether it is administered incombination with other agents, timing of administration, the tissue towhich it is administered, and the physical delivery system in which itis carried.

As used herein, the terms “treatment” or “treating” a disease ordisorder refers to a method of reducing, delaying or ameliorating such acondition before or after it has occurred. Treatment may be directed atone or more effects or symptoms of a disease and/or the underlyingpathology. Treatment is aimed to obtain beneficial or desired resultsincluding, but not limited to, therapeutic benefit and/or a prophylacticbenefit. By therapeutic benefit is meant eradication or amelioration ofthe underlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient can still be afflicted with the underlying disorder. Forprophylactic benefit, the pharmaceutical compounds and/or compositionscan be administered to a patient at risk of developing a particulardisease, or to a patient reporting one or more of the physiologicalsymptoms of a disease, even though a diagnosis of this disease may nothave been made. The treatment can be any reduction and can be, but isnot limited to, the complete ablation of the disease or the symptoms ofthe disease. As compared with an equivalent untreated control, suchreduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%,60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

As used herein, the term “therapeutic effect” refers to a therapeuticbenefit and/or a prophylactic benefit as described herein. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

As used herein, the term “pharmaceutically acceptable ester” refers toesters that hydrolyze in vivo and include those that break down readilyin the human body to leave the parent compound or a salt thereof. Suchesters can act as a prodrug as defined herein. Pharmaceuticallyacceptable esters include, but are not limited to, alkyl, alkenyl,alkynyl, aryl, aralkyl, and cycloalkyl esters of acidic groups,including, but not limited to, carboxylic acids, phosphoric acids,phosphinic acids, sulfinic acids, sulfonic acids and boronic acids.Examples of esters include formates, acetates, propionates, butyrates,acrylates and ethylsuccinates. The esters can be formed with a hydroxyor carboxylic acid group of the parent compound.

As used herein, the term “pharmaceutically acceptable enol ethers”include, but are not limited to, derivatives of formula —C═C(OR) where Rcan be selected from alkyl, alkenyl, alkynyl, aryl, aralkyl andcycloalkyl. Pharmaceutically acceptable enol esters include, but are notlimited to, derivatives of formula —C═C(OC(O)R) where R can be selectedfrom hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl and cycloalkyl.

As used herein, a “pharmaceutically acceptable form” of a disclosedcompound includes, but is not limited to, pharmaceutically acceptablesalts, hydrates, esters, solvates, isomers, prodrugs, and isotopicallylabeled derivatives of disclosed compounds. In one embodiment, a“pharmaceutically acceptable form” includes, but is not limited to,pharmaceutically acceptable salts, isomers, prodrugs and isotopicallylabeled derivatives of disclosed compounds. In some embodiments, a“pharmaceutically acceptable form” includes, but is not limited to,pharmaceutically acceptable salts, stereoisomers, prodrugs andisotopically labeled derivatives of disclosed compounds.

In certain embodiments, the pharmaceutically acceptable form is apharmaceutically acceptable salt. As used herein, the term“pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of subjects without undue toxicity, irritation,allergic response and the like, and are commensurate with a reasonablebenefit/risk ratio. Pharmaceutically acceptable salts are well known inthe art. For example, Berge et al. describes pharmaceutically acceptablesalts in detail in J. Pharmaceutical Sciences (1977) 66:1-19.Pharmaceutically acceptable salts of the compounds provided hereininclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchioric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. In some embodiments, organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, lactic acid, trifluoracetic acid,maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, andthe like.

The salts can be prepared in situ during the isolation and purificationof the disclosed compounds, or separately, such as by reacting the freebase or free acid of a parent compound with a suitable base or acid,respectively. Pharmaceutically acceptable salts derived from appropriatebases include alkali metal, alkaline earth metal, ammonium andN⁺(C₁₋₄alkyl)⁴ salts. Representative alkali or alkaline earth metalsalts include sodium, lithium, potassium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate and aryl sulfonate. Organic bases fromwhich salts can be derived include, for example, primary, secondary, andtertiary amines, substituted amines, including naturally occurringsubstituted amines, cyclic amines, basic ion exchange resins, and thelike, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. In some embodiments,the pharmaceutically acceptable base addition salt can be chosen fromammonium, potassium, sodium, calcium, and magnesium salts.

In certain embodiments, the pharmaceutically acceptable form is a“solvate” (e.g., a hydrate). As used herein, the term “solvate” refersto compounds that further include a stoichiometric or non-stoichiometricamount of solvent bound by non-covalent intermolecular forces. Thesolvate can be of a disclosed compound or a pharmaceutically acceptablesalt thereof. Where the solvent is water, the solvate is a “hydrate”.Pharmaceutically acceptable solvates and hydrates are complexes that,for example, can include 1 to about 100, or 1 to about 10, or 1 to about2, about 3 or about 4, solvent or water molecules. It will be understoodthat the term “compound” as used herein encompasses the compound andsolvates of the compound, as well as mixtures thereof.

In certain embodiments, the pharmaceutically acceptable form is aprodrug. As used herein, the term “prodrug” (or “pro-drug”) refers tocompounds that are transformed in vivo to yield a disclosed compound ora pharmaceutically acceptable form of the compound. A prodrug can beinactive when administered to a subject, but is converted in vivo to anactive compound, for example, by hydrolysis (e.g., hydrolysis in blood).In certain cases, a prodrug has improved physical and/or deliveryproperties over the parent compound. Prodrugs can increase thebioavailability of the compound when administered to a subject (e.g., bypermitting enhanced absorption into the blood following oraladministration) or which enhance delivery to a biological compartment ofinterest (e.g., the brain or lymphatic system) relative to the parentcompound. Exemplary prodrugs include derivatives of a disclosed compoundwith enhanced aqueous solubility or active transport through the gutmembrane, relative to the parent compound.

The prodrug compound often offers advantages of solubility, tissuecompatibility or delayed release in a mammalian organism (see, e.g.,Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam). A discussion of prodrugs is provided in Higuchi, T., et al.,“Pro-drugs as Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14,and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche,American Pharmaceutical Association and Pergamon Press, 1987, both ofwhich are incorporated in full by reference herein. Exemplary advantagesof a prodrug can include, but are not limited to, its physicalproperties, such as enhanced water solubility for parenteraladministration at physiological pH compared to the parent compound, orit can enhance absorption from the digestive tract, or it can enhancedrug stability for long-term storage.

As used herein, the term “pharmaceutically acceptable” excipient,carrier, or diluent refers to a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject pharmaceutical agent from one organ, or portionof the body, to another organ, or portion of the body. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the patient. Someexamples of materials which can serve as pharmaceutically-acceptablecarriers include: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations. Wetting agents, emulsifiers and lubricants,such as sodium lauryl sulfate, magnesium stearate, and polyethyleneoxide-polypropylene oxide copolymer as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

Compounds of the present invention are, subsequent to their preparation,preferably isolated and purified to obtain a composition containing anamount by weight equal to or greater than 95% (“substantially pure”),which is then used or formulated as described herein. In certainembodiments, the compounds of the present invention are more than 99%pure.

Solvates and polymorphs of the compounds of the invention are alsocontemplated herein. Solvates of the compounds of the present inventioninclude, for example, hydrates.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. When a range of values is listed, it isintended to encompass each value and sub-range within the range. Forexample “C₁₋₆ alkyl” is intended to encompass, C₁, C₂, C₃, C₄, C₅, C₆,C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆, C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄,C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

As used herein, the term “alkyl” refers to a straight or branchedhydrocarbon chain radical consisting solely of carbon and hydrogenatoms, containing no unsaturation, having from one to ten carbon atoms(e.g., C₁₋₁₀ alkyl). Whenever it appears herein, a numerical range suchas “1 to 10” refers to each integer in the given range; e.g., “1 to 10carbon atoms” means that the alkyl group can consist of 1 carbon atom, 2carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms,although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated. In some embodiments,“alkyl” can be a C₁₋₆ alkyl group. In some embodiments, alkyl groupshave 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms. Representativesaturated straight chain alkyls include, but are not limited to,-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; whilesaturated branched alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl,3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl,2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl,2,3-dimethylbutyl, and the like. The alkyl is attached to the parentmolecule by a single bond. Unless stated otherwise in the specification,an alkyl group is optionally substituted by one or more of substituentswhich independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy,alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino,imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy,haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio,thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl,sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea,—Si(R^(a))₃, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R_(a),—C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, —N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or 2), —P(═O)(R^(a))(R^(a)), or—O—P(═O)(OR^(a))₂ where each R^(a) is independently hydrogen, alkyl,haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl,and each of these moieties can be optionally substituted as definedherein. In a non-limiting embodiment, a substituted alkyl can beselected from fluoromethyl, difluoromethyl, trifluoromethyl,2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl,3-hydroxypropyl, benzyl, and phenethyl.

As used herein, the term “alkoxy” refers to the group —O-alkyl,including from 1 to 10 carbon atoms (C₁₋₁₀) of a straight, branched,saturated cyclic configuration and combinations thereof, attached to theparent molecular structure through an oxygen. Examples include methoxy,ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, pentoxy, cyclopropyloxy,cyclohexyloxy and the like. “Lower alkoxy” refers to alkoxy groupscontaining one to six carbons. In some embodiments, C₁₋₃ alkoxy is analkoxy group which encompasses both straight and branched chain alkylsof from 1 to 3 carbon atoms. Unless stated otherwise in thespecification, an alkoxy group can be optionally substituted by one ormore substituents which independently include: acyl, alkyl, alkenyl,alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino,amido, amidino, imino, azide, carbonate, carbamate, carbonyl,heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy,cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio,alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate,phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl,sulfonate, urea, —Si(R^(a))₃, —OR^(a), —SR^(a), —OC(O)—R^(a),—N(R^(a))₂, —C(O)R_(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,—N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or2), —P(═O)(R^(a))(R^(a)), or —P(═O)(OR^(a))₂ where each R^(a) isindependently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl,aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl orheteroarylalkyl, and each of these moieties can be optionallysubstituted as defined herein.

As used herein, the terms “aromatic” or “aryl” refer to a radical with 6to 14 ring atoms (e.g., C₆₋₁₄ aromatic or C₆₋₁₄ aryl) which has at leastone ring having a conjugated pi electron system which is carbocyclic(e.g., phenyl, fluorenyl, and naphthyl). In some embodiments, the arylis a C₆₋₁₀ aryl group. For example, bivalent radicals formed fromsubstituted benzene derivatives and having the free valences at ringatoms are named as substituted phenylene radicals. In other embodiments,bivalent radicals derived from univalent polycyclic hydrocarbon radicalswhose names end in “-yl” by removal of one hydrogen atom from the carbonatom with the free valence are named by adding “-idene” to the name ofthe corresponding univalent radical, e.g., a naphthyl group with twopoints of attachment is termed naphthylidene. Whenever it appearsherein, a numerical range such as “6 to 14 aryl” refers to each integerin the given range; e.g., “6 to 14 ring atoms” means that the aryl groupcan consist of 6 ring atoms, 7 ring atoms, etc., up to and including 14ring atoms. The term includes monocyclic or fused-ring polycyclic (i.e.,rings which share adjacent pairs of ring atoms) groups. Polycyclic arylgroups include bicycles, tricycles, tetracycles, and the like. In amulti-ring group, only one ring is required to be aromatic, so groupssuch as indanyl are encompassed by the aryl definition. Non-limitingexamples of aryl groups include phenyl, phenalenyl, naphthalenyl,tetrahydronaphthyl, phenanthrenyl, anthracenyl, fluorenyl, indolyl,indanyl, and the like. Unless stated otherwise in the specification, anaryl moiety can be optionally substituted by one or more substituentswhich independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy,alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino,imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy,haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio,thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl,sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea,—Si(R^(a))₃, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R_(a),—C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, —N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or 2), —P(═O)(R^(a))(R^(a)), or—O—P(═O)(OR^(a))₂ where each R^(a) is independently hydrogen, alkyl,haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl,and each of these moieties can be optionally substituted as definedherein.

As used herein, the terms “cycloalkyl” and “carbocyclyl” each refers toa monocyclic or polycyclic radical that contains only carbon andhydrogen, and can be saturated or partially unsaturated. Partiallyunsaturated cycloalkyl groups can be termed “cycloalkenyl” if thecarbocycle contains at least one double bond, or “cycloalkynyl” if thecarbocycle contains at least one triple bond. Cycloalkyl groups includegroups having from 3 to 13 ring atoms (i.e., C₃₋₁₃ cycloalkyl). Wheneverit appears herein, a numerical range such as “3 to 10” refers to eachinteger in the given range; e.g., “3 to 13 carbon atoms” means that thecycloalkyl group can consist of 3 carbon atoms, 4 carbon atoms, 5 carbonatoms, etc., up to and including 13 carbon atoms. The term “cycloalkyl”also includes bridged and spiro-fused cyclic structures containing noheteroatoms. The term also includes monocyclic or fused-ring polycyclic(i.e., rings which share adjacent pairs of ring atoms) groups.Polycyclic aryl groups include bicycles, tricycles, tetracycles, and thelike. In some embodiments, “cycloalkyl” can be a C₃₋₈ cycloalkylradical. In some embodiments, “cycloalkyl” can be a C₃₋₅ cycloalkylradical. Illustrative examples of cycloalkyl groups include, but are notlimited to the following moieties: C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclobutyl (C₄), cyclopentyl (C₅),cyclopentenyl (C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl(C₆) and the like. Examples of C₃₋₇ carbocyclyl groups include norbornyl(C₇). Examples of C₃₋₈ carbocyclyl groups include the aforementionedC₃₋₇ carbocyclyl groups as well as cycloheptyl (C₇), cycloheptadienyl(C₇), cycloheptatrienyl (C₇), cyclooctyl (C₈), bicyclo[2.2.1]heptanyl,bicyclo[2.2.2]octanyl, and the like. Examples of C₃₋₁₃ carbocyclylgroups include the aforementioned C₃₋₈ carbocyclyl groups as well asoctahydro-1H indenyl, decahydronaphthalenyl, spiro[4.5]decanyl and thelike. Unless stated otherwise in the specification, a cycloalkyl groupcan be optionally substituted by one or more substituents whichindependently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl,cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide,carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy,haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio,thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl,sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea,—Si(R^(a))₃, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R_(a),—C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, —N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or 2), —P(═O)(R^(a))(R^(a)), or—O—P(═O)(OR^(a))₂ where each R^(a) is independently hydrogen, alkyl,haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl,and each of these moieties can be optionally substituted as definedherein. The terms “cycloalkenyl” and “cycloalkynyl” mirror the abovedescription of “cycloalkyl” wherein the prefix “alk” is replaced with“alken” or “alkyn” respectively, and the parent “alkenyl” or “alkynyl”terms are as described herein. For example, a cycloalkenyl group canhave 3 to 13 ring atoms, such as 5 to 8 ring atoms. In some embodiments,a cycloalkynyl group can have 5 to 13 ring atoms.

As used herein, the term “halide”, “halo”, or, alternatively, “halogen”means fluoro, chioro, bromo or iodo. The terms “haloalkyl,”“haloalkenyl,” “haloalkynyl” and “haloalkoxy” include alkyl, alkenyl,alkynyl and alkoxy structures that are substituted with one or more halogroups or with combinations thereof. For example, the terms“fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxygroups, respectively, in which the halo is fluorine, such as, but notlimited to, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, and the like. Each of the alkyl, alkenyl,alkynyl and alkoxy groups are as defined herein and can be optionallyfurther substituted as defined herein.

As used herein, the term “heteroalkyl” refers to an alkyl radical, whichhave one or more skeletal chain atoms selected from an atom other thancarbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinationsthereof. A numerical range can be given, e.g., C₁₋₄ heteroalkyl whichrefers to the chain length in total, which in this example is 4 atomslong. For example, a —CH₂OCH₂CH₃ radical is referred to as a “C₄”heteroalkyl, which includes the heteroatom center in the atom chainlength description. Connection to the parent molecular structure can bethrough either a heteroatom or a carbon in the heteroalkyl chain. Forexample, an N-containing heteroalkyl moiety refers to a group in whichat least one of the skeletal atoms is a nitrogen atom. One or moreheteroatom(s) in the heteroalkyl radical can be optionally oxidized. Oneor more nitrogen atoms, if present, can also be optionally quaternized.For example, heteroalkyl also includes skeletal chains substituted withone or more nitrogen oxide (—O—) substituents. Exemplary heteroalkylgroups include, without limitation, ethers such as methoxyethanyl(—CH₂CH₂OCH₃), ethoxymethanyl (—CH₂OCH₂CH₃), (methoxymethoxy)ethanyl(—CH₂CH₂OCH₂OCH₃), (methoxymethoxy) methanyl (—CH₂OCH₂OCH₃) and(methoxyethoxy)methanyl (—CH₂OCH₂CH₂OCH₃) and the like; amines such as(—CH₂CH₂NHCH₃, —CH₂CH₂N(CH₃)₂, —CH₂NHCH₂CH₃, —CH₂N(CH₂CH₃)(CH₃)) and thelike.

As used herein, the term “heteroaryl” or, alternatively,“heteroaromatic” refers to a refers to a radical of a 5-18 memberedmonocyclic or polycyclic (e.g., bicyclic, tricyclic, tetracyclic and thelike) aromatic ring system (e.g., having 6, 10 or 14 π electrons sharedin a cyclic array) having ring carbon atoms and 1-6 ring heteroatomsprovided in the aromatic ring system, wherein each heteroatom isindependently selected from nitrogen, oxygen, phosphorous and sulfur(“5-18 membered heteroaryl”). Heteroaryl polycyclic ring systems caninclude one or more heteroatoms in one or both rings. Whenever itappears herein, a numerical range such as “5 to 18” refers to eachinteger in the given range; e.g., “5 to 18 ring atoms” means that theheteroaryl group can consist of 5 ring atoms, 6 ring atoms, etc., up toand including 18 ring atoms. In some instances, a heteroaryl can have 5to 14 ring atoms. In some embodiments, the heteroaryl has, for example,bivalent radicals derived from univalent heteroaryl radicals whose namesend in “-yl” by removal of one hydrogen atom from the atom with the freevalence are named by adding “-ene” to the name of the correspondingunivalent radical, e.g., a pyridyl group with two points of attachmentis a pyridylene.

For example, an N-containing “heteroaromatic” or “heteroaryl” moietyrefers to an aromatic group in which at least one of the skeletal atomsof the ring is a nitrogen atom. One or more heteroatom(s) in theheteroaryl radical can be optionally oxidized. One or more nitrogenatoms, if present, can also be optionally quaternized. Heteroaryl alsoincludes ring systems substituted with one or more nitrogen oxide (—O—)substituents, such as pyridinyl N-oxides. The heteroaryl is attached tothe parent molecular structure through any atom of the ring(s).

“Heteroaryl” also includes ring systems wherein the heteroaryl ring, asdefined above, is fused with one or more aryl groups wherein the pointof attachment to the parent molecular structure is either on the aryl oron the heteroaryl ring, or wherein the heteroaryl ring, as definedabove, is fused with one or more cycloalkyl or heterocycyl groupswherein the point of attachment to the parent molecular structure is onthe heteroaryl ring. For polycyclic heteroaryl groups wherein one ringdoes not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl andthe like), the point of attachment to the parent molecular structure canbe on either ring, i.e., either the ring bearing a heteroatom (e.g.,2-indolyl) or the ring that does not contain a heteroatom (e.g.,5-indolyl). In some embodiments, a heteroaryl group is a 5-10 memberedaromatic ring system having ring carbon atoms and 1-4 ring heteroatomsprovided in the aromatic ring system, wherein each heteroatom isindependently selected from nitrogen, oxygen, phosphorous, and sulfur(“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group isa 5-8 membered aromatic ring system having ring carbon atoms and 1-4ring heteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen, phosphorous,and sulfur (“5-8 membered heteroaryl”). In some embodiments, aheteroaryl group is a 5-6 membered aromatic ring system having ringcarbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, phosphorous, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, phosphorous, and sulfur. In someembodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatomsselected from nitrogen, oxygen, phosphorous, and sulfur. In someembodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selectedfrom nitrogen, oxygen, phosphorous, and sulfur.

Examples of heteroaryls include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl,benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, benzo[b][1,4] oxazinyl, 1,4-benzodioxanyl,benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl,benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl,benzopyranonyl, benzofurazanyl, benzothiazolyl, benzothienyl(benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl,5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5Hbenzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo [3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocycloocta[d] pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl,indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl,isoquinolyl, indolizinyl, isoxazolyl,5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl,pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl,pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl,quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo [4,5]thieno[2,3-d]pyrimdinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl,thiazolyl, thiadiazolyl, thiapyranyl, triazolyl, tetrazolyl, triazinyl,thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e., thienyl). Unless stated otherwisein the specification, a heteroaryl moiety can be optionally substitutedby one or more substituents which independently include: acyl, alkyl,alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy,amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl,heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy,cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio,alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate,phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl,sulfonate, urea, —Si(R^(a))₃, —OR^(a), —SR^(a), —OC(O)—R^(a),—N(R^(a))₂, —C(O)R_(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,—N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or2), -P(═O)(R^(a))(R^(a)), or —O—P(═O)(OR^(a))₂ where each R^(a) isindependently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl,aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl orheteroarylalkyl, and each of these moieties can be optionallysubstituted as defined herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the unexpected discovery of novel, orallyavailable, selective and potent inhibitors of mutated IDH 1 and/or IDH 2proteins. The compounds disclosed here form irreversible covalent bondor reversible binding interactions with mutant IDH 1 and/or IDH 2protein and effectively inhibit their respective alpha hydroxylneoactivity.

Several IDH inhibitors are currently being studied including GSK321 andAG-221. These compounds reportedly bind to IDH1, IDH2, or both IDH1 andIDH2 in a reversible manner.

The reported reversible inhibitors have shown less than optimal potency,selectivity and exposure time.

In contrast, in certain aspects the present invention provides a unique,irreversible inhibition strategy that affords significant improvedpotency, selectivity and exposure time presumably due to the covalentbonding and a prolonged pharmacodynamics.

Additionally, in certain aspectsm the present invention provides anoptimized , reversible inhibition compounds that affords significantimproved potency, selectivity and exposure time.

As disclosed herein below, cysteine 379 of IDH1 (cys379:fmtkdlaacikglpnvqrsd) is selected as a target for irreversible covalentbond formation. The cysteine, which has proximity of 3.8 Å to thehydroxyl group on GSK321 (FIG. 1), is discovered to be a suitable targetfor irreversible covalent bond formation.

As disclosed herein below, cysteine 269 of IDH1 (cys269:seggfiwacknydgdvqsds) is also selected as a target for irreversiblecovalent bond formation. The cysteine, which has proximity of 8 Å to thephenyl ring on GSK321 (FIG. 1), is discovered to be a suitable targetfor irreversible covalent bond formation.

The new strategy disclosed herein allows the translation of a reversibleIDH1/2 inhibitor to an irreversible or reversible inhibitor byincorporation an appropriate functional group, such as an electrophilicgroup (e.g., acrylamide) to form a covalent bond (e.g., S—C bond) orbinding interaction with the targetable amino acid (e.g., cysteine).

A series of novel compounds are specifically designed, synthesized andtested. These compounds, each bearing an electrophilic group suitablefor reaction with the target cysteine to form an irreversible covalentbond or a reversible binding interaction.

Advantages of the approach disclosed herein include sustained targetinhibition, which can be achieved with only transient exposure of thetarget to the inhibitor. This approach reduces the need to achievepharmacological properties that would allow for sustained drug levels invivo.

In one aspect, the invention generally relates to a compound having thestructural formula of (I):

wherein,

-   -   each of Ring A, Ring B and Ring C is independently a 5- or        6-membered cyclic or bicyclic alkyl, heteroalkyl, aryl, or        heteroaryl ring;    -   each R_(W) is independently a hydrogen, a halide, OH, NH₂,        alkyl, alkyl amine, alkyl amide, alkoxy, or an electrophilic        group R_(E);    -   R₁ is H, halide, alkyl amine, methoxy, hydroxy or a C₁-C₃ alkyl        group;    -   X is CH₂, NH, O, S or a single bond;    -   Y is CH₂, NH, O or S; and    -   Z is CH₂, NH, O or S,        or a pharmaceutical acceptable form thereof.

In certain embodiments, each of Ring A, Ring B and Ring C isindependently a 5,6-bicyclic aryl or heteroaryl, or a 6,6-bicyclic arylor heteroaryl.

In certain embodiments, the compound has the structural formula of (II).

In certain embodiments, the compound has the structural formula of(I-A).

In certain embodiments, the compound has the structural formula of(II-A).

In certain embodiments, the compound has the structural formula of(I-B).

In certain embodiments, the compound has the structural formula of(II-B).

In certain embodiments, the compound has the structural formula of(I-C):

In certain embodiments, the compound has the structural formula of(II-C):

In certain embodiments, each of Ring A and Ring B is a 6-membered arylgroup and Ring C is a 5-membered aryl group.

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

In certain embodiments, the compound has the structural formula of:

R_(E) may be any suitable electrophilic group. Exemplary R_(E) groupsinclude the R_(E) groups found in the exemplary compounds of theinvention, as disclosed herein.

In certain embodiments, R_(E) comprises an acrylamide group. In certainembodiments, R_(E) is an acrylamide group.

In certain embodiments, R_(E) comprises

In certain embodiments, R_(E) is

In certain embodiments, R_(E) comprises

In certain embodiments, R_(E) comprises

In certain embodiments, R_(E) comprises

Exemplary compounds of the invention include:

Exemplary compounds of the invention also include:

Exemplary compounds of the invention further include:

A unique aspect of the present invention is the IDH inhibitors disclosedherein form one or more covalent bond with the target IDH. The type ofactual covalent bond formed between the inhibitor and the IDH isdependent on the inhibitor's warhead group (c.g., —R_(E)) and thecorresponding reactive group on the IDH. Below are illustrative examplesinvolving a cysteine reacting with two electrophilic groups viacysteine's —SH moiety.

In another aspect, the invention generally relates to a pharmaceuticalcomposition comprising a compound having the structural formula of (I):

wherein,

-   -   each of Ring A, Ring B and Ring C is independently a 5- or        6-membered cyclic or bicyclic alkyl, heteroalkyl, aryl, or        heteroaryl ring;    -   each R_(W) is independently —R_(E) or a hydrogen, a halide, OH,        NH₂, alkyl, alkyl amine, alkyl amide, alkoxy; R_(E) herein is an        electrophilic group.    -   R₁ is H, halide, alkyl amine, methoxy, hydroxy or a C₁-C₃ alkyl        group;    -   X is CH₂, NH, O, S or a single bond;    -   Y is CH₂, NH, O or S; and    -   Z is CH₂, NH, O or S,        or a pharmaceutically acceptable form thereof, effective to        treat, prevent, or reduce one or more cancers, or a related        disease or disorder thereof, in a mammal, including a human, and        a pharmaceutically acceptable excipient, carrier, or diluent.

In certain embodiments, each of Ring A, Ring B and Ring C isindependently a 5,6-bicyclic aryl or heteroaryl, or a 6,6-bicyclic arylor heteroaryl.

In certain embodiments, the compound has the structural formula (II).

In yet another aspect, the invention generally relates to apharmaceutical composition comprising a compound disclosed herein.

In yet another aspect, the invention generally relates to a unit dosageform comprising a pharmaceutical composition disclosed herein.

In yet another aspect, the invention generally relates to a method fortreating, reducing, or preventing a disease or disorder, comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising a compound having the structural formula of (I):

wherein,

-   -   each of Ring A, Ring B and Ring C is independently a 5- or        6-membered cyclic or bicyclic alkyl, heteroalkyl, aryl, or        heteroaryl ring;    -   each R_(W) is independently —R_(E) or a hydrogen, a halide, OH,        NH₂, alkyl, alkyl amine, alkyl amide, alkoxy; R_(E) herein is an        electrophilic group.    -   R₁ is H, halide, alkyl amine, methoxy, hydroxy or a C₁-C₃ alkyl        group;    -   X is CH₂, NH, O, S or a single bond;    -   Y is CH₂, NH, O or S; and    -   Z is CH₂, NH, O or S,        or a pharmaceutically acceptable form thereof, effective to        treat, prevent, or reduce one or more cancers, or a related        disease or disorder thereof, in a mammal, including a human, and        a pharmaceutically acceptable excipient, carrier, or diluent.

In certain embodiments, each of Ring A, Ring B and Ring C isindependently a 5,6-bicyclic aryl or heteroaryl, or a 6,6-bicyclic arylor heteroaryl.

In certain embodiments of the method, the compound has the structuralformula.

In certain embodiments, the one or more cancers comprise a blood canceror a hematologic malignance. In certain embodiments, the one or morecancers are selected from B-acute lymphoblastic leukemias, B-acutelymphoblastic leukemias, chronic myelomonocytic leukemia, Acutemyelogenous leukemia, lymphoma, myelodysplasia syndrome,myeloproliferative neoplasms and myeloproliferative neoplasms.

Any appropriate route of administration can be employed, for example,parenteral, intravenous, subcutaneous, intramuscular, intraventricular,intracorporeal, intraperitoneal, rectal, or oral administration. Mostsuitable means of administration for a particular patient will depend onthe nature and severity of the disease or condition being treated or thenature of the therapy being used and on the nature of the activecompound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the compoundsdescribed herein or derivatives thereof are admixed with at least oneinert customary excipient (or carrier) such as sodium citrate ordicalcium phosphate or (i) fillers or extenders, as for example,starches, lactose, sucrose, glucose, mannitol, and silicic acid, (ii)binders, as for example, carboxymethylcellulose, alignates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, (iii) humectants, as forexample, glycerol, (iv) disintegrating agents, as for example,agar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain complex silicates, and sodium carbonate, (v) solution retarders,as for example, paraffin, (vi) absorption accelerators, as for example,quaternary ammonium compounds, (vii) wetting agents, as for example,cetyl alcohol, and glycerol monostearate, (viii) adsorbents, as forexample, kaolin and bentonite, and (ix) lubricants, as for example,talc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, or mixtures thereof. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like. Solid dosage forms such as tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells, such as entericcoatings and others known in the art.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents, and emulsifiers, such as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil, sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid estersof sorbitan, or mixtures of these substances, and the like. Besides suchinert diluents, the composition can also include additional agents, suchas wetting, emulsifying, suspending, sweetening, flavoring, or perfumingagents.

Materials, compositions, and components disclosed herein can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. It is understoodthat when combinations, subsets, interactions, groups, etc. of thesematerials are disclosed that while specific reference of each variousindividual and collective combinations and permutations of thesecompounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a method is disclosedand discussed and a number of modifications that can be made to a numberof molecules including in the method are discussed, each and everycombination and permutation of the method, and the modifications thatare possible are specifically contemplated unless specifically indicatedto the contrary. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed. This concept applies to allaspects of this disclosure including, but not limited to, steps inmethods using the disclosed compositions. Thus, if there are a varietyof additional steps that can be performed, it is understood that each ofthese additional steps can be performed with any specific method stepsor combination of method steps of the disclosed methods, and that eachsuch combination or subset of combinations is specifically contemplatedand should be considered disclosed.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios arecontemplated by the present invention. Those of ordinary skill in theart will readily appreciate that analogous ratios are contemplated formore complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic methods well known in the art, and subsequent recoveryof the pure enantiomers.

EXAMPLES The synthesis of1-(3-amino-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-1)

Tert-butyl 3-methyl-4-oxopiperidine-1-carboxylate (2) To a solution oftert-butyl 4-oxopiperidine-1-carboxylate (1) (100 g, 0.5 mol) inanhydrous THF (1000, mL) was added LDA (300 mL) dropwise at −78° C.under nitrogen atmosphere. After 1 h, MeI (70.9 g, 0.5 mol) was added tothe same temperature and then the mixture was stirred at roomtemperature for another 3 h. The mixture was quenched with saturatedNH₄Cl solution and was portioned between ethyl acetate and water. Theorganic layer was washed with brine, dried over Na₂SO₄, and concentratedto give orange oil. The residue was purified by silica gel columnchromatography (petroleum ether:ethyl acetate=20:1) to afford thecompound as light yellow oil (30.4 g, 28%). MS m/z 214.23[M+H]⁺.

Tert-butyl3-(2-ethoxy-2-oxoacetyl)-5-methyl-4-oxopiperidine-1-carboxylate (3) To asolution of tert-butyl 3-methyl-4-oxopiperidine-1-carboxylate (2) (30.4g, 0.14 mol) in anhydrous THF (350 mL) was added LDA (2M, 84 mL)dropwise at −78° C. under nitrogen atmosphere. The mixture was stirredat −78° C. for 1 h and diethyl oxalate (20.46 g, 0.14 mol) was addedinto the reaction solution. The resulting mixture was allowed to warm upto room temperature and stirred for 2 h. The mixture was neutralizedwith 1 N HCl and extracted with ethyl acetate. The organic layer waswashed with brine, dried over Na₂SO₄, concentrated under reducedpressure and the residue was purified by silica gel chromatography(petroleum ether:ethyl acetate=10:1) to afford the compound as a yellowoil (20 g, 45.6%). MS m/z 314.15[M+H]⁺.

5-(tert-butyl) 3-ethyl7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(4) To a solution of tert-butyl3-(2-ethoxy-2-oxoacetyl)-5-methyl-4-oxopiperidine-1-carboxylate (3) (20g, 63.8 mmol) in acetic acid (80 mL) was added hydrazine hydrate (9.6g). After 2 h, the reaction mixture was concentrated under reducedpressure and the residue was diluted with water and saturated aqueousNaHCO₃ and then extracted with ethyl acetate. The combined organiclayers were dried over Na₂SO₄, filtered, concentrated under reducedpressure and dried to provide compound (4) (16 g, 81%) as a pale yellowsolid. MS m/z 310.17[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 13.35 (s, 1H), 4.42(d, J=16.3 Hz, 2H), 4.30-4.23 (m, 2H), 4.03 (q, J=7.1 Hz, 1H), 3.75 (s,1H), 2.94 (s, 2H), 1.42 (s, 9H), 1.28 (m, J=7.0 Hz, 3H), 1.18 (m, 3H).

5-(tert-butyl) 3-ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) To a solution of 5-(tert-butyl) 3-ethyl7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(4) (1.5 g, 4.85mmol) in dry THF (20 mL) was added NaH (60% wtdispersion in mineral oil, 0.23 g, 5.82mmol) with stirring undernitrogen. After 1 h, 4-(bromomethyl)-1-fluoro-2-nitrobenzene (1.13 g,4.85mmol) was added and the reaction mixture was stirred 4 h undernitrogen. The reaction mixture was quenched with water and the pH wasadjusted to 6 with 1 N HCl. The mixture was diluted with brine andextracted with ethyl acetate. The combined extracts were dried overNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas purified by silica gel chromatography (petroleum ether:ethylacetate=5:1) to provide 5-(tert-butyl) 3-ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(0.91 g, 40.6%) as light yellow solid. MS m/z 463.39[M+H]⁺.

Ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylatehydrochloride (7) To a round-bottom flask containing 5-(tert-butyl)3-ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) (0.91 g, 2 mmol) was added HCl (4 N in ethyl acetate, 5 mL). Theresulting clear solution was stirred at room temperature for 3 h. Thereaction mixture was concentrated under reduced pressure and theresulting residue was dried to provide ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylatehydrochloride (0.78 g, 100%) as a yellow solid.

1H-pyrrole-2-carbonyl chloride (8A) To a solution of1H-pyrrole-2-carboxylic acid (8)(0.3 g) in DCM (15 mL) was added oxalylchloride (0.28 mL) and a drop of DMF at 0° C. The resulting clearsolution was stirred at room temperature for 3 h. The reaction mixturewas concentrated under reduced pressure. The solvent was distilled offunder reduced pressure from the reaction mixture, and the residue wassubjected to an azeotropic distillation treatment with toluene. Thecrude product of 1H-2-pyrrolecarbonyl chloride (0.35 g) was obtained asa brown crystalline product which was used directly in the next step.

Ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) To a solution of ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylatehydrochloride (7) (0.8 g, 2.5 mmol) in DCM (10 mL) was added DIEA (0.65g, 6.25 mmol) and 1H-pyrrole-2-carbonyl chloride (0.31 g, 3 mmol) at 0°C. for 10 min. The reaction mixture was diluted with water and saturatedaqueous NaHCO₃ and extracted with two portions of DCM. The organic layerwas washed with brine, dried over Na₂SO₄, concentrated under reducedpressure and the residue was purified by silica gel chromatography(petroleum ether:ethyl acetate=3:1) to afford the compound as a yellowsolid (0.8 g, 73%). MS m/z 456.30[M+H]⁺.

1-(4-fluoro-3-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) To a solution of ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) (0.8 g, 1.76 mmol) in 1,4-dioxane (5 mL) was added 5 N LiOH (5 mL).The clear solution was stirred at room temperature for 1 h. The reactionmixture was added water and pH was adjusted to 6 with 1 N HCl. Theresulting precipitate was collected by filtration and dried on alyophilizer overnight to provide1-(4-fluoro-3-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (0.7 g, 92%) as a white solid. MS m/z 428.19[M+H]⁺.

5-((1H-pyrrol-2-yl)methyl)-1-(4-fluoro-3-nitrobenzyl)-7-methyl-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(4-fluoro-3-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.32 g, 0.75 mmol) in DMF (5 mL) were added m-toluidine (0.1g, 0.9 mmol), HATU (0.43 g, 1.125 mmol) and DIEA (0.2 g, 1.5 mmol). Themixture was stirred at room temperature under nitrogen. After 1 h, thereaction mixture was added water and the resulting precipitate wascollected by filtration and dried on a lyophilizer overnight to providecompound (12)(0.36 g, 95%) as a white solid. MS m/z 517.20[M+H]⁺.

1-(3-amino-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of5-((1H-pyrrol-2-yl)methyl)-1-(4-fluoro-3-nitrobenzyl)-7-methyl-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.36 g, 0.7 mmol) in ethyl acetate/methanol (4/1)(10 mL) was addedstannous chloride (1.12 g, 5.6 mmol). The resulting mixture was heatedat 80° C. overnight. The reaction mixture was added DCM and the pH wasadjusted to 8 with saturated aqueous NaHCO₃ and extracted with DCM. Theorganic layer was washed with brine, dried over Na₂SO₄, concentratedunder reduced pressure and the residue was purified by silica gelchromatography (DCM:MeOH=50:1) to afford the compound as a white solid(0.3 g, 88%). MS m/z 487.28[M+H]⁺.

1-(3-amino-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-1) To a solution of1-(3-acrylamido-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.3 g, 0.62 mmol) in DCM (10 mL) was added TEA (0.19 g, 1.86 mmol)and acryloyl chloride (0.17 g, 1.86 mmol) at 0° C. for 10 min. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃ andextracted with DCM. The organic layer was washed with brine, dried overNa₂SO₄, concentrated under reduced pressure and the residue was purifiedby silica gel chromatography (DCM:MeOH=20:1) to afford the compound as awhite solid (86 mg, 26%). MS m/z 541.3[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ11.53 (s, 1H), 9.98 (s, 1H), 9.84 (s, 1H), 7.98 (d, J=5.9 Hz, 1H), 7.68(s, 1H), 7.57 (d, J=8.3 Hz, 1H), 7.26 (m, 1H), 7.19 (t, J=8.0 Hz, 1H),6.95 (m, 1H), 6.91 (m, 2H), 6.60 (m, 2H), 6.26 (dd, J=17.0, 1.9 Hz, 1H),6.16 (m, 1H), 5.77 (dd, J=10.4, 2.2 Hz, 1H), 5.45 (q, J=16.4 Hz, 2H),5.30 (d, J=16.4 Hz, 1H), 4.65 (br, 1H), 4.36 (d, J=11.1 Hz, 1H), 3.41(m, 1H), 3.20 (m, 1H), 2.29 (s, 3H), 1.13 (t, J=8.9 Hz, 3H).

The synthesis of1-(3-(acrylamidomethyl)-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-2)

5-(tert-butyl)3-ethyl1-(3-cyano-4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) To a solution of 5-(tert-butyl) 3-ethyl7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(4) (0.8 g, 2.6 mmol) in dry THF (20 mL) was added NaH(60% wt dispersionin mineral oil, 0.2 g, 5 mmol) with stirring under nitrogen. After 1 h,5-(bromomethyl)-2-fluorobenzonitrile (0.7 g, 3.3 mmol) was added and thereaction mixture was stirred 4 h under nitrogen. The reaction mixturewas quenched with water and the pH was adjusted to 6 with 1 N HCl. Themixture was diluted with brine and extracted with ethyl acetate. Thecombined extracts were dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The residue was purified by silica gelchromatography (petroleum ether:ethyl acetate=5:1) to provide5-(tert-butyl) 3-ethyl1-(3-cyano-4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(1.4 g, 95%) as a light yellow solid. MS m/z 442.2[M+H]⁺.

ethyl1-(3-cyano-4-fluorobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(7) To a round-bottom flask containing 5-(tert-butyl) 3-ethyl1-(3-cyano-4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) (1.4 g, 2mmol) was added HCl (4 N in ethyl acetate, 5 mL). Theresulting clear solution was stirred at room temperature for 3 h. Thereaction mixture was quenched with water and the pH was adjusted to 7with NaHCO₃. The mixture was diluted with brine and extracted with DCM.The combined extracts were dried over Na₂SO₄, filtered and concentratedunder reduced pressure and the resulting residue was dried to provideethyl1-(3-cyano-4-fluorobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(0.8 g, 60%) as a yellow solid. MS m/z 343.15[M+H]⁺.

ethyl1-(3-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) To a solution of ethyl1-(3-cyano-4-fluorobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(7) (0.8 g, 2.5 mmol) in DCM (10 mL) was added DIEA(0.65 g, 6.25mmol)and 1H-pyrrole-2-carbonyl chloride (0.31 g, 3mmol) at 0° C. for 10 min.The reaction mixture was diluted with water and saturated aqueous NaHCO₃and extracted with two portions of DCM. The organic layer was washedwith brine, dried over Na₂SO₄, concentrated under reduced pressure andthe residue was purified by silica gel chromatography (petroleumether:ethyl acetate=2:1) to afford the compound as a yellow solid (0.8g, 70%). MS m/z 436.17[M+H]⁺.

1-(3-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) To a solution ofethyl1-(3-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) (0.8 g, 1.84 mmol) in 1,4-dioxane (5 mL) was added 5 N LiOH (5 mL).The clear solution was stirred at room temperature for 1 h. The reactionmixture was added water and pH was adjusted to 6 with 1 N HCl. Theresulting precipitate was collected by filtration and dried on alyophilizer overnight to provide1-(3-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (0.7 g, 89%) as a white solid. MS m/z 408.14[M+H]⁺.

1-(3-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(3-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.72 g, 1.75 mmol) in DMF (5 mL) were added m-toluidine (0.4g, 3.7 mmol), HATU (0.8 g, 2.1 mmol) and DIEA (0.4 g, 3 mmol). Themixture was stirred at room temperature under nitrogen. After 1 h, thereaction mixture was added water and the resulting precipitate wascollected by filtration and dried on a lyophilizer overnight to providecompound (12)(0.85 g, 99%) as a white solid. MS m/z 497.20[M+H]⁺.

1-(3-(aminomethyl)-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of1-(3-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.36 g, 0.7 mmol) in methanol (10 mL) was added Hydrochloric acid(2 mL) and Pd/C (0.2 g). The resulting mixture was stirred at roomtemperature overnight under H2. The organic layer was concentrated underreduced pressure and the residue was purified by silica gelchromatography (DCM:MeOH=50:1) to afford the compound as a white solid(0.3 g, 88%). MS m/z 501.23[M+H]⁺.

1-(3-(acrylamidomethyl)-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-2) To a solution of1-(3-(aminomethyl)-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.2 g, 0.4 mmol) in DCM(10 ml) was added TEA (0.13 g, 1.2 mmol)and acryloyl chloride (0.04 g, 0.44 mmol) at 0° C. for 10 min. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃ andextracted with DCM. The organic layer was washed with brine, dried overNa₂SO₄, concentrated under reduced pressure and the residue was purifiedby silica gel chromatography (DCM:MeOH=20:1) to afford the compound as awhite solid (112 mg, 43%). MS m/z 555.24[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ11.53 (s, 1H), 9.81 (s, 1H), 8.60 (m, 1H), 7.68 (s, 1H), 7.56 (d, J=7.9Hz, 1H), 7.24-7.08 (m, 4H), 6.91 (m, 2H), 6.59 (m, 1H), 6.27-6.13 (m,2H), 6.04 (dd, J=17.1, 2.1 Hz, 1H), 5.51 (dd, J=10.1, 2.1 Hz, 1H), 5.41(q, J=16.2 Hz, 2H), 5.26 (d, J=16.5 Hz, 1H), 4.63 (br, 1H), 4.35 (m,3H), 3.30 (s, 1H), 3.16-3.07 (m, 1H), 2.29 (s, 3H), 1.07 (d, J=6.8 Hz,3H).

The synthesis of1-(3-acrylamido-4-fluorobenzyl)-N-(3((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-3)

5-((1H-pyrrol-2-yl)methyl)-1-(4-fluoro-3-nitrobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(4-fluoro-3-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.32 g, 0.75 mmol) in DMF (5 mL) were added(S)-1-(3-aminophenyl)ethan-1-ol (11)(0.12 g, 0.9 mmol), HATU (0.43 g,1.125 mmol) and DIEA (0.2 g, 1.5 mmol). The mixture was stirred at roomtemperature under nitrogen. After 1 h, the reaction mixture was addedwith water and the resulting precipitate was collected by filtration anddried on a lyophilizer overnight to provide compound (12) (0.38 g, 93%)as a white solid. MS m/z 547.22[M+H]⁺.

1-(3-amino-4-fluorobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of5-((1H-pyrrol-2-yl)methyl)-1-(4-fluoro-3-nitrobenzyl)-N-(3-((5)-1-hydroxyethyl)phenyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.38 g, 0.7 mmol) in EA/methanol (4/1)(10 mL) was added stannouschloride (1.1 g, 5.6 mmol). The resulting mixture was heated at 80° C.overnight. The reaction mixture was added DCM and the pH was adjusted to8 with saturated aqueous NaHCO₃ and extracted with DCM. The organiclayer was washed with brine, dried over Na₂SO₄, concentrated underreduced pressure and the residue was purified by silica gelchromatography (DCM:MeOH=50:1) to afford the compound (13) as a whitesolid (0.325 g, 90%). MS m/z 517.28[M+H]⁺.

1-(3-acrylamido-4-fluorobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-3) To a solution of1-(3-amino-4-fluorobenzyl)-N-(3-((5)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13)(0.16 g, 0.31 mmol) in DCM (5 mL) was added TEA (63 mg, 0.62 mmol)and acryloyl chloride (42.2 g, 46.5 mmol) at 0° C. for 10 min. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃ andextracted with two portions of DCM. The organic layer was washed withbrine, dried over Na₂SO₄, concentrated under reduced pressure and theresidue was purified by silica gel chromatography (DCM:MeOH=20:1) toafford the compound as a white solid (35 mg, 20%). MS m/z 571.17[M+H]⁺.¹H NMR (400 MHz, DMSO) δ 11.53 (s, 1H), 9.98 (s, 1H), 9.88 (s, 1H), 7.97(d, J=6.0 Hz, 1H), 7.83 (s, 1H), 7.63 (m, 1H), 7.30-7.18 (m, 2H), 7.05(d, J=7.6 Hz, 1H), 6.97 (m, 1H), 6.91 (m, 1H), 6.66-6.53 (m, 2H), 6.26(dd, J=16.8, 2.0 Hz, 1H), 6.17 (m, 1H), 5.75 (m, 1H), 5.43 (q, J=16.0Hz, 2H), 5.30 (d, J=16.4 Hz, 1H), 5.13 (d, J=4.1 Hz, 1H), 4.74-4.59 (m,2H), 4.36 (d, J=11.3 Hz, 1H), 3.50-3.41 (m, 1H), 3.25-3.11 (m, 1H), 1.31(d, J=6.4 Hz, 3H), 1.12 (d, J=6.8 Hz, 3H).

The synthesis ofN-(3-acrylamidophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-4)

5-(tert-butyl) 3-ethyl1-(4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) To a solution of 5-(tert-butyl) 3-ethyl7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(4) (2 g, 6.46 mmol) in dry THF (30 mL) was added NaH (60% wt dispersionin mineral oil, 0.31 g, 7.75 mmol) with stirring under nitrogen. After 1h, 1-(bromomethyl)-4-fluorobenzene (1.5 g, 6.46 mmol) was added and thereaction mixture was stirred under nitrogen for 4 h. The reactionmixture was quenched with water and the pH was adjusted to 6 with 1 NHCl. The mixture was diluted with brine and extracted with ethylacetate. The combined extracts were dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by silicagel chromatography (petroleum ether:ethyl acetate=5:1) to afford5-(tert-butyl) 3-ethyl1-(4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(1.05 g, 34%) as a light yellow solid. MS m/z 418.2[M+H]⁺.

Ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylatehydrochloride (7) To a round-bottom flask containing 5-(tert-butyl)3-ethyl1-(4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) (2.52 g, 6.04 mmol) was added HCl (4 N in ethyl acetate, 20 mL). Theresulting clear solution was stirred at room temperature for 3 h. Thereaction mixture was concentrated under reduced pressure and theresulting residue was dried under high vacuum to afford ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylatehydrochloride (2.13 g, 100%) as a yellow solid.

Ethyl1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) To a solution of ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylatehydrochloride (7)(0.35 g, 1 mmol) in DMF (10 mL) were added1H-pyrrole-2-carboxylic acid (0.12 g, 1.1 mmol), HATU (0.57 g, 1.5 mmol)and DIEA (0.39 g, 3 mmol). The mixture was stirred at room temperatureunder nitrogen. After 1 h, the reaction mixture was added with water andthe resulting precipitate was collected by filtration and dried on alyophilizer overnight to afford compound (9) (0.29 g, 70%) as a whitesolid. MS m/z 411.32[M+H]⁺.

1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) To a solution of ethyl1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) (0.2 g, 0.49 mmol) in MeOH (3 mL) was added 5N LiOH (1 mL). Theclear solution was stirred at room temperature for 1 h. The reactionmixture was added water and pH was adjusted to 6 with 1 N HCl. Theresulting precipitate was collected by filtration and dried on alyophilizer overnight to afford1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (0.1 g, 53.5%) as a white solid. MS m/z 383.13[M+H]⁺.

N-(3-aminophenyl)acrylamide (13) To a solution of benzene-1,3-diamine(11) (5.4 g, 50 mmol) in DCM (100) was added DIEA (13 g, 100 mmol) andacryloyl chloride (4.5 g, 50 mmol) at 0° C. for 10 min. The reactionmixture was diluted with water and saturated aqueous NaHCO₃ andextracted with two portions of DCM. The organic layer was washed withbrine, dried over Na₂SO₄, concentrated under reduced pressure and theresidue was purified by silica gel chromatography (DCM:MeOH=20:1) toafford the compound as a brown solid (2.4 g, 30%). MS m/z 163.08[M+H]⁺.N-(3-acrylamidophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-4) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (38 mg, 0.1 mmol) in DMF (2 mL) were addedN-(3-aminophenyl)acrylamide (18 mg, 0.11 mmol), HATU (57 mg, 0.15 mmol)and DIEA (26 mg, 0.2 mmol). The mixture was stirred at room temperatureunder nitrogen. After 1 h, the reaction mixture was diluted with waterand extracted with DCM. The organic layer was washed with brine, driedover Na₂SO₄, concentrated under reduced pressure and the residue waspurified by silica gel chromatography (DCM:MeOH=20:1) to afford thecompound as a white solid (32 mg, 60.1%). MS m/z 527.85[M+H]⁺. ¹H NMR(400 MHz, DMSO) δ 11.53 (s, 1H), 10.16 (s, 1H), 9.99 (s, 1H), 8.23 (m,1H), 7.54 (m, 1H), 7.37 (m, 1H), 7.31-7.12 (m, 5H), 6.91 (m, 1H), 6.58(m, 1H), 6.47 (m, 1H), 6.26 (dd, J=16.8, 2.0 Hz, 1H), 6.18 (m, 1H), 5.73(dd, J=16.0, 2.0 Hz, 1H), 5.45 (q, J=16.4 Hz, 2H), 5.32 (d, J=15.6Hz,1H), 4.78-4.58 (m, 1H), 4.35 (d, J=13.2 Hz, 1H), 3.38 (m, 1H),3.22-3.10(m, 1H), 1.09 (d, J=6.8 Hz, 3H).

The synthesis ofN-(3-(2-chloroacetamido)phenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-5)

N-(3-aminophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.1 g, 0.26 mmol) in DMF (5 mL) were addedbenzene-1,3-diamine (30.3 mg, 0.28 mmol), HATU (0.15 g, 0.39 mmol) andDIEA (67.2 g, 0.52 mmol). The mixture was stirred at room temperatureunder nitrogen. After 1 h, the reaction mixture was added water and theresulting precipitate was collected by filtration and dried on alyophilizer overnight to afford compound (12) (88 mg, 72%) as a whitesolid. MS m/z 473.2[M+H]⁺.

N-(3-(2-chloroacetamido)phenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-5) To a solution ofN-(3-aminophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (50 mg, 0.106 mmol) in DCM (5 mL) was added TEA (22 mg, 0.212 mmol)and 2-chloroacetyl chloride (24 mg, 0.212 mmol) at 0° C. for 10 min. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃ andextracted with two portions of DCM. The organic layer was washed withbrine, dried over Na₂SO₄, concentrated under reduced pressure and theresidue was purified by silica gel chromatography (DCM:MeOH =20:1) toafford the compound as a white solid (20 mg, 35%). MS m/z 549.17 [M+H]⁺.¹H NMR (400 MHz, DMSO) δ 11.52 (s, 1H), 10.36 (s, 1H), 10.02 (s, 1H),8.18 (s, 1H), 7.42 (m, 2H), 7.31-7.14 (m, 5H), 6.91 (m, 1H), 6.58 (m,1H), 6.18 (m, 1H), 5.45 (q, J=16.2 Hz, 2H), 5.31 (d, J=16.7 Hz, 1H),4.69 (br, 1H), 4.35 (d, J=13.6 Hz, 1H), 4.26 (s, 2H), 3.47-3.34 (m, 1H),3.17 (d, J=5.2 Hz, 1H), 1.09 (d, J=6.9 Hz, 3H).

The synthesis ofN-(1-acryloylpiperidin-3-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-6)

Tert-butyl3-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)piperidine-1-carboxylate(12) To a solution of1-(4-fluoro-3-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylic acid (10) (0.1 g, 0.26 mmol) in DMF (5 mL)were added tert-butyl 3-aminopiperidine-1-carboxylate (56 mg, 0.28mmol), HATU (0.15 g, 0.39 mmol) and DIEA (67 mg, 0.52 mmol). The mixturewas stirred at room temperature under nitrogen. After 1 h, the reactionmixture was added with water and the resulting precipitate was collectedby filtration and dried on a lyophilizer overnight to afford compound(12) (0.1 g, 68%)as a white solid. MS m/z 565.2[M+H]⁺.

1-(4-fluorobenzyl)-7-methyl-N-(piperidin-3-yl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamidehydrochloride (13) To a round-bottom flask containingtert-butyl3-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)piperidine-1-carboxylate(12) (0.1 g, 0.18 mmol) was added HCl (4 N in ethyl acetate, 5 mL). Theresulting solution was stirred at room temperature for 3 h. The reactionmixture was concentrated under reduced pressure and the resultingresidue was dried to constant weight under high vacuum to afford1-(4-fluorobenzyl)-7-methyl-N-(piperidin-3-yl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamidehydrochloride (90 mg, 100%) as a yellow solid.

N-(1-acryloylpiperidin-3-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-6) To a solution of1-(4-fluorobenzyl)-7-methyl-N-(piperidin-3-yl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamidehydrochloride (13) (90 mg, 0.18 mmol) in DCM(5 ml) was added DIEA (70mg, 0.54 mmol) and acryloyl chloride (32 mg, 0.36 mmol) at 0° C. for 10min. The reaction mixture was diluted with water and saturated aqueousNaHCO₃ and extracted with DCM. The organic layer was washed with brine,dried over Na₂SO₄, concentrated under reduced pressure and the residuewas purified by silica gel chromatography (DCM:MeOH=20:1) to afford thecompound as a white solid (33.6 mg, 36%). MS m/z 519.6 [M+H]⁺. ¹H NMR(400 MHz, DMSO) δ 11.51 (s, 1H), 7.94 (br, 1H), 7.19 (m, 4H), 6.90 (m,1H), 6.76 (br, 1H), 6.56 (s, 1H), 6.17 (m, 1H), 6.08 (d, J=16.0 Hz, 1H),5.66 (dd, J=10.5, 2.3 Hz, 1H), 5.38 (q, J=16.4 Hz, 2H), 5.24 (d, J=16.3Hz, 1H), 4.61 (m, 1H), 4.31 (m, 1H), 4.12 (m, 1H), 3.89 (m, 1H), 3.78(s, 1H), 3.44-3.33 (m, 1H), 3.30-2.88 (m, 3H), 2.78 (m, 1H), 1.83 (m,1H), 1.69 (m, 2H), 1.06 (d, J=6.3 Hz, 3H).

The synthesis of1-(2-acrylamido-4-fluorobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-7)

5-(tert-butyl) 3-ethyl1-(4-fluoro-2-nitrobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) To a solution of 5-(tert-butyl) 3-ethyl7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(4) (1.24 g, 4 mmol) in dry THF (20 mL) was added NaH (60% wt dispersionin mineral oil, 0.2 g, 4.8 mmol) with stirring under nitrogen. After 1h, 1-(bromomethyl)-4-fluoro-2-nitrobenzene (0.94 g, 4 mmol) was addedand the reaction mixture was stirred 4h under nitrogen. The reactionmixture was quenched with water and pH adjusted to 6 with 1N HCl. Themixture was diluted with brine and extracted with ethyl acetate. Thecombined extracts were dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The residue was purified by silica gelchromatography (petroleum ether:ethyl acetate=5:1) to afford5-(tert-butyl) 3-ethyl1-(4-fluoro-2-nitrobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(1 g, 54%) as a light yellow solid. MS m/z 463.17[M+H]⁺.

Ethyl1-(4-fluoro-2-nitrobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylatehydrochloride (7) To a round-bottom flask containing 5-(tert-butyl)3-ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) (1 g) was added HCl (4 N in EA, 10 mL). The resulting clear solutionwas stirred at room temperature for 3 h. The reaction mixture wasconcentrated under reduced pressure and the resulting residue was driedto under high vacuum to afford ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylatehydrochloride (0.85 g, 100%) as a yellow solid.

Ethyl1-(4-fluoro-2-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) To a solution of ethyl1-(4-fluoro-2-nitrobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylatehydrochloride (7) (0.85 g, 2.1 mmol) in DCM (10 mL) was added DIEA(0.67g, 5.2 mmol) and 1H-pyrrole-2-carbonyl chloride (8A) (0.6 g, 2.5 mmol)at 0° C. for 10 min. The reaction mixture was diluted with water andsaturated aqueous NaHCO₃ and extracted with DCM. The organic layer waswashed with brine, dried over Na₂SO₄, concentrated under reducedpressure and the residue was purified by silica gel chromatography(petroleum ether:ethyl acetate=3:1) to afford the compound as a yellowsolid (0.76 g, 80%). MS m/z 456.37[M+H]⁺.

1-(4-fluoro-2-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) To a solution of ethyl1-(4-fluoro-2-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) (0.1 g, 0.22 mmol) in MeOH (3 mL) was added 5N LiOH (1 mL). Theclear solution was stirred at room temperature for 1 h. The reactionmixture was added water and pH was adjusted to 6 with 1 N HCl. Theresulting precipitate was collected by filtration and dried on alyophilizer overnight to afford1-(4-fluoro-2-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (70mg, 74.5%) as a white solid. MS m/z 428.39[M+H]⁺.

1-(4-fluoro-2-nitrobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(4-fluoro-2-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (70 mg, 0.16 mmol) in DMF (2 mL) were added(S)-1-(3-aminophenyl)ethan-1-ol (25 mg, 0.18 mmol), HATU (92 mg, 0.24mmol) and DIEA (42 mg, 0.32 mmol). The mixture was stirred at roomtemperature under nitrogen. After 1 h, the reaction mixture was addedwater and the resulting precipitate was collected by filtration anddried on a lyophilizer overnight to afford compound (12)(74 mg, 85%) asa white solid. MS m/z 547.48[M+H]⁺.

1-(2-amino-4-fluorobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of5-((1H-pyrrol-2-yl)methyl)-1-(4-fluoro-2-nitrobenzyl)-N-(3-((5)-1-hydroxyethyl)phenyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.4 g, 0.73 mmol) in ethyl acetate/methanol (4/1)(10 mL) was addedstannous chloride (1.11 g, 5.86 mmol). The resulting mixture was heatedat 80° C. overnight. The reaction mixture was added DCM and the p Hadjusted to ˜8 with saturated aqueous NaHCO₃ and extracted with DCM. Theorganic layer was washed with brine, dried over Na₂SO₄, concentratedunder reduced pressure and the residue was purified by silica gelchromatography (DCM:MeOH=50:1) to afford the compound as a white solid(0.36 g, 95.5%). MS m/z 517.1[M+H]⁺.

1-(2-acrylamido-4-fluorobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-7) To a solution of1-(2-amino-4-fluorobenzyl)-N-(3-((5)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.36 g, 0.7 mmol) in DCM (5 mL) was added DIEA (0.18 g, 1.4 mmol)and acryloyl chloride (95 mg, 1.05 mmol) at 0° C. for 10 min. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃ andextracted with DCM. The organic layer was washed with brine, dried overNa₂SO₄, concentrated under reduced pressure and the residue was purifiedby silica gel chromatography (DCM:MeOH=20:1) to afford the compound as awhite solid (60 mg, 15%). MS m/z 571.4[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ11.54 (s, 1H), 10.19 (s, 1H), 9.95 (s, 1H), 7.86 (s, 1H), 7.76 (m, 1H),7.43 (m, 1H), 7.08 (m, 1H), 7.03 (m, 1H), 6.91 (m, 1H), 6.80 (m, 1H),6.60 (s, 1H), 6.40-6.33 (m, 1H), 6.29 (s, 1H), 6.20 (m, 1H), 5.96 (dd,J=10.4, 1.6 Hz,1H), 5.85 (m, 1H), 5.80 (m, 1H), 5.41 (q, J=16.4 Hz, 2H),5.29 (d, J=16.5 Hz, 1H), 4.67 (d, J=14.7 Hz, 1H), 4.32 (d, J=13.1 Hz,1H), 3.42 (m, 1H), 3.17 (m, 1H), 3.13 (s, 1H), 1.51 (d, J=6.6 Hz, 3H),1.08 (d, J=6.9 Hz, 3H).

The synthesis of1-(2-(acrylamidomethyl)-4-fluorobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-8)

2-(bromomethyl)-5-fluorobenzonitrile (5) To a solution of5-fluoro-2-methylbenzonitrile (5a)(5 g, 37 mmol) in CCl₄(50 mL) wasadded NBS (7.3 g, 41 mmol) and AIBN (0.6 g, 3.7 mmol). The resultingmixture was heated at 80° C. overnight. The reaction mixtureconcentrated under reduced pressure and the residue was purified bysilica gel chromatography (petroleum ether/ethylacetate=5:1) to affordthe compound as a white solid (2.8 g, 35%). MS m/z 214.96[M+H]⁺.

5-(tert-butyl)3-ethyll-(2-cyano-4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) To a solution of 5-(tert-butyl) 3-ethyl7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(4) (0.8 g, 2.6 mmol) in dry THF (20 mL) was added NaH (60% wtdispersion in mineral oil, 0.2 g, 5 mmol) with stirring under nitrogen.After 1 h, 2-(bromomethyl)-5-fluorobenzonitrile (0.7 g, 3.3 mmol) wasadded and the reaction mixture was stirred 4 h under nitrogen. Thereaction mixture was quenched with water and the pH was adjusted to 6with 1 N HCl. The mixture was diluted with brine and extracted withethyl acetate. The combined extracts were dried over Na₂SO₄, filteredand concentrated under reduced pressure. The residue was purified bysilica gel chromatography (petroleum ether:ethyl acetate=5:1) to afford5-(tert-butyl) 3-ethyl1-(2-cyano-4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(1.4 g, 95%) as a light yellow solid. MS m/z 443.2[M+H]⁺.

Ethyl1-(2-cyano-4-fluorobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(7) To a round-bottom flask containing 5-(tert-butyl) 3-ethyl1-(2-cyano-4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) (1.4 g, 2 mmol) was added HCl (4 N in ethyl acetate, 5 mL). Theresulting clear solution was stirred at room temperature for 3 h. Thereaction mixture was quenched with water and pH was adjusted to 7 withNaHCO₃. The mixture was diluted with brine and extracted with DCM. Thecombined extracts were dried over Na₂SO₄, filtered and concentratedunder reduced pressure and the resulting residue was dried under highvacuum to afford ethyl1-(2-cyano-4-fluorobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(0.8 g, 60%) as a yellow solid. MS m/z 343.1[M+H]⁺.

Ethyl1-(2-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) To a solution of ethyl1-(2-cyano-4-fluorobenzyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(7) (0.8 g, 2.5 mmol) in DCM (10 mL) was added DIEA (0.65 g, 6.25 mmol)and 1H-pyrrole-2-carbonyl chloride (0.31 g, 3 mmol) at 0° C. for 10 min.The reaction mixture was diluted with water and saturated aqueous NaHCO₃and extracted with two portions of DCM. The organic layer was washedwith brine, dried over Na₂SO₄, concentrated under reduced pressure andthe residue was purified by silica gel chromatography (petroleumether:ethyl acetate=2:1) to afford the compound as a yellow solid (0.8g, 70%). MS m/z 436.1[M+H]⁺.

1-(2-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) To a solution of ethyl1-(2-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) (0.8 g, 1.84 mmol) in 1,4-dioxane (5 mL) was added 5 N LiOH (5 mL).The clear solution was stirred at room temperature for lh. The reactionmixture was added water and pH was adjusted o 6 with 1 N HCl. Theresulting precipitate was collected by filtration and dried on alyophilizer overnight to afford1-(2-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (0.7 g, 89%) as a white solid. MS m/z 408.1[M+H]⁺.

1-(2-cyano-4-fluorobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(2-cyano-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.72 g, 1.75 mmol) in DMF (5 mL) were added(S)-1-(3-aminophenyl)ethan-1-ol (0.4 g, 3.5 mmol), HATU (0.8 g, 2.1mmol) and DIEA (0.4 g, 3 mmol). The mixture was stirred at roomtemperature under nitrogen. After 1 h, the reaction mixture was addedwith water and the resulting precipitate was collected by filtration anddried on a lyophilizer overnight to afford compound (12)(0.85 g, 99%) asa white solid. MS m/z 527[M+H]⁺.

1-(2-(aminomethyl)-4-fluorobenzyl)-N-(3-((S)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of1-(2-cyano-4-fluorobenzyl)-N-(3-((5)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.2 g, 0.4 mmol) in methanol (10 mL) was added hydrochloric acid(2 mL) and Pd/C (0.2 g). The resulting mixture was stirred at roomtemperature overnight under H2. The organic layer was concentrated underreduced pressure and the residue was purified by silica gelchromatography (DCM:MeOH=50:1) to afford the compound as a white solid(0.06 g, 23%). MS m/z 531[M+H]⁺.

1-(2-(acrylamidomethyl)-4-fluorobenzyl)-N-(3-((S)1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-8) To a solution of1-(2-(aminomethyl)-4-fluorobenzyl)-N-(3-((5)-1-hydroxyethyl)phenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.06 g, 0.13 mmol) in DCM (10 mL) was added TEA (0.05 g, 0.4 mmol)and acryloyl chloride (0.015 g, 0.15 mmol) at 0° C. for 10 min. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃ andextracted with two portions of DCM. The organic layer was washed withbrine, dried over Na₂SO₄, concentrated under reduced pressure and theresidue was purified by silica gel chromatography (DCM:MeOH=20:1) toafford the compound as a white solid (31 mg, 46%). MS m/z 585.25[M+H]⁺.¹H NMR (400 MHz, DMSO) 611.54 (s, 1H), 9.87 (s, 1H), 8.82 (s, 1H), 7.82(s, 1H), 7.63 (m, 1H), 7.25 (m, 1H), 7.11 (m, 1H), 7.03 (m, 2H), 6.92(m, 1H), 6.70 (m,1H), 6.59 (m, 1H), 6.37 (m, 1H), 6.23-6.12 (m, 2H),5.67 (dd, J=10.2, 2.1 Hz, 1H), 5.56 (q, J=16.4 Hz, 2H), 5.34 (d, J=16.6Hz, 1H), 5.14 (s, 1H), 4.75-4.45 (m, 4H), 4.37 (d, J=11.8 Hz, 1H), 3.39(m, 1H), 3.19 (m, 1H), 1.31 (d, J=6.4 Hz, 3H), 1.06 (dd, J=14.1, 6.9 Hz,3H).

Synthesis ofN-(1-acryloylpyrrolidin-3-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-9)

tert-butyl-3-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)pyrrolidine-1-carboxylate(12) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.3 g, 0.8 mmol) in DMF (5 mL) were added tert-butyl3-aminopyrrolidine-1-carboxylate (0.16 g, 0.9 mmol) HATU (0.45 g, 1.2mmol) and DIEA (0.2 g, 1.6 mmol). The mixture was stirred at roomtemperature under nitrogen. After 1 h, the reaction mixture was addedwith water and the resulting precipitate was collected by filtration anddried to afford compound (12) (150 mg, 72%) as a white solid. MS m/z551.25[M+H]⁺.

1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(pyrrolidin-3-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a round-bottom flask containingtert-butyl-3-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)pyrrolidine-1-carboxylate(12) (0.15 g, 0.27mmol) was added HCl (4 N in ethyl acetate, 5 mL). Theresulting clear solution was stirred at room temperature. The reactionmixture was concentrated under reduced pressure and the resultingresidue was dried to afford the product 13 (130 mg, 100%) as a yellowsolid.

N-(1-acryloylpyrrolidin-3-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-9) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(pyrrolidin-3-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (130 mg, 0.28 mmol) in DCM (5 mL) was added TEA (90 mg, 0.54 mmol)and acryloyl chloride (30 mg, 0.36 mmol) at 0° C. The reaction mixturewas diluted with water and saturated aqueous NaHCO₃ and extracted withDCM. The organic layer was washed with brine, dried over Na₂SO₄,concentrated under reduced pressure and the residue was purified bysilica gel chromatography to afford the compound as a white solid (50mg, 36%). MS m/z 505 [M+H]⁺. 1H NMR (400 MHz, DMSO) δ 11.53 (s, 1H),8.35 (m, 1H), 7.18 (d, J=7.4 Hz, 4H), 6.91 (s, 1H), 6.62 (m, 2H), 6.22(m, 1H), 6.08 (m, 1H), 5.65 (m, 1H), 5.38 (q, J=16.2 Hz, 2H), 5.24 (d,J=16.4 Hz, 1H), 4.60 (m, 1H), 4.47-4.40 (m, 1H), 4.32 (d, J=12.7 Hz,1H), 3.88-3.46 (m, 3H), 3.39 (m, 2H), 3.12 (m, 1H), 2.22-1.90 (m, 2H),1.05 (d, J=6.8 Hz, 3H).

The synthesis ofN-(1-acryloylazetidin-3-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-10)

3-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)azetidine-1-carboxylate(12) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.1 g, 0.26 mmol) in DMF (5mL) were added tert-butyl3-aminoazetidine-1-carboxylate (38 mg, 0.28 mmol), HATU (0.15 g, 0.39mmol) and DIEA (67.2 g, 0.52 mmol). The mixture was stirred at roomtemperature under nitrogen. After 1 h, the reaction mixture was addedwith water and the resulting precipitate was collected by filtration anddried to afford compound (12)(95 mg, 72%) as a white solid. MS m/z537[M+H]⁺.

N-(azetidin-3-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a round-bottom flask containingtert-butyl-3-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)azetidine-1-carboxylate(12) (0.95 g, 0.18 mmol) was added with HCl (4 N in ethyl acetate, 5mL). The resulting clear solution was stirred at room temperature andthen the reaction mixture was concentrated under reduced pressure andthe resulting residue was dried under high vacuum to afford the product13 (90 mg, 100%) as a yellow solid.

N-(1-acryloylazetidin-3-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-10) To a solution ofN-(azetidin-3-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (90 mg, 0.106 mmol) in DCM (5 mL) was added TEA (22 mg, 0.212 mmol)and 2-chloroacetyl chloride (30 mg, 0.212 mmol) at 0° C. The reactionmixture was diluted with water and saturated aqueous NaHCO₃ andextracted with DCM. The organic layer was washed with brine, dried overNa₂SO₄, concentrated under reduced pressure and the residue was purifiedby silica gel chromatography to afford the compound as a white solid (42mg, 46%). MS m/z 491[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.52 (s, 1H), 8.85(d, J=7.4 Hz, 1H), 7.20 (m, 4H), 6.90 (s, 1H), 6.57 (s, 1H), 6.30 (m,1H), 6.16 (m, 1H), 6.09 (dd, J=17.0, 2.3 Hz, 1H), 5.66 (dd, J=10.3, 2.2Hz, 1H), 5.38 (q, J=16.2 Hz , 2H), 5.23 (d, J=16.5 Hz, 1H), 4.76-4.64(m, 1H), 4.58 (m, 1H), 4.46 (m, 1H), 4.32 (d, J=12.5 Hz, 1H), 4.21 (m,1H), 4.14 (t, J=9.1 Hz, 1H), 4.04-3.93 (m, 1H), 3.35 (br, 1H), 3.15 (m,1H), 1.06 (d, J=6.7 Hz, 3H).

The synthesis ofN-(5-acrylamidothiophen-2-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-19)

1-(4-fluorobenzyl)-7-methyl-N-(5-nitrothiophen-2-yl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.1 g, 0.26 mmol) in DMF (5 mL) were added5-nitrothiophen-2-amine (48 mg, 0.28 mmol), HATU (0.15 g, 0.39 mmol) andDIEA (0.067 g, 0.52 mmol). The mixture was stirred at room temperatureunder nitrogen. After 1 h, the reaction mixture was added with water andthe resulting precipitate was collected by filtration and dried toafford compound (12) (120 mg, 82%) as a white solid. MS m/z509.13[M+H]⁺.

N-(5-aminothiophen-2-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of1-(4-fluorobenzyl)-7-methyl-N-(5-nitrothiophen-2-yl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.12 g, 0.25 mmol) in methanol (10 mL) was added hydrochloric acid(2 mL) and Pd/C (0.2 g). The resulting mixture was stirred at roomtemperature under H2. The organic layer was concentrated under reducedpressure and the residue was purified by silica gel chromatography toafford the compound as a white solid (0.11 g, 88%). MS m/z 479[M+H]⁺.

N-(5-acrylamidothiophen-2-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-19) To a solution ofN-(5-aminothiophen-2-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (110 mg, 0.206 mmol) in DCM (5 mL) was added TEA (44 mg, 0.42 mmol)and 2-chloroacetyl chloride (48 mg, 0.4 mmol) at 0° C. The reactionmixture was diluted with water and saturated aqueous NaHCO₃ andextracted with DCM. The organic layer was washed with brine, dried overNa₂SO₄, concentrated under reduced pressure and the residue was purifiedby silica gel chromatography to afford the compound as a white solid (50mg, 45%). MS m/z 533.17[M+H]⁺. 1H NMR (400 MHz, DMSO) δ 11.54 (s, 1H),11.24 (s, 1H), 11.12 (s, 1H), 7.30 (m, 4H), 6.92 (s, 1H), 6.77 (d, J=4.4Hz, 1H), 6.63 (s, 1H), 6.49 (d, J=4.1 Hz, 1H), 6.39 (m, 1H), 6.28 (m,2H), 5.76 (d, J=10.1 Hz, 1H), 5.45 (q, J=16.4 Hz, 2H), 5.33 (d, J=16.5Hz, 1H), 4.69 (d, J=13.4 Hz, 1H), 4.35 (d, J=11.7 Hz, 1H), 3.42 (br,1H), 3.18 (m, 1H), 1.08 (d, J=6.8 Hz, 3H)

The synthesis ofN-(3-acrylamidocyclobutyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-20)

(3-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)cyclobutyl)carbamate(12) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.3 g, 0.8 mmol) in DMF (5 mL) were added tert-butyl(3-aminocyclobutyl)carbamate (0.16 g, 0.9 mmol), HATU (0.45 g, 1.2 mmol)and DIEA (0.2 g, 1.6 mmol). The mixture was stirred at room temperatureunder nitrogen. After 1 h, the reaction mixture was added water and theresulting precipitate was collected by filtration and dried bylyophilizer overnight to provide compound (12) (150 mg, 72%) as a whitesolid. MS m/z 551.25[M+H]⁺.

N-(3-aminocyclobutyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a round-bottom flask containingtert-butyl-3-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)cyclobutyl)carbamate(12) (0.15 g, 0.27 mmol) was added HCl (4 N in ethyl acetate, 5 mL). Theresulting clear solution was stirred at room temperature. The reactionmixture was concentrated under reduced pressure and the resultingresidue was dried to constant weight under high vacuum to afford theproduct (130 mg, 100%) as a yellow solid.

N-(3-acrylamidocyclobutyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-20) To a solution ofN-(3-aminocyclobutyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (130 mg, 0.28 mmol) in DCM (5 mL) was added TEA (90 mg, 0.54 mmol)and acryloyl chloride (30 mg, 0.36 mmol) at 0° C. The reaction mixturewas diluted with water and saturated aqueous NaHCO₃ and extracted withDCM. The organic layer was washed with brine, dried over Na₂SO₄,concentrated under reduced pressure and the residue was purified bysilica gel chromatography to afford the compound as a white solid (50mg, 36%). MS m/z 505 [M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.51 (s, 1H),8.51 (d, J=10.0 Hz, 1H), 8.35 (d, J=10.0 Hz, 1H), 7.28 (m, 4H), 6.90 (s,1H), 6.53 (s, 1H), 6.28 (m, 2H), 6.07 (m, 1H), 5.57 (d, J=10.0 Hz, 1H),5.38 (m, 2H), 5.23 (d, J=16.5 Hz, 1H), 4.73-3.85 (m, 4H), 3.36 (m, 1H),3.15-3.05 (m, 1H), 2.48-2.35 (m, 2H), 2.27-2.01 (m, 2H), 1.06 (d, J=6.8Hz, 3H)

The synthesis ofN-(4-acrylamidophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-25)

N-(3-aminophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.1 g, 0.26 mmol) in DMF (5 mL) were addedbenzene-1,4-diamine (28 mg, 0.28 mmol), HATU (0.15 g, 0.39 mmol) andDIEA (67.2 g, 0.52 mmol). The mixture was stirred at room temperatureunder nitrogen. After 1 h, the reaction mixture was added with water andthe resulting precipitate was collected by filtration and dried toprovide compound (12) (105 mg, 78%) as a white solid. MS m/z 473.20[M+H]⁺.

N-(4-acrylamidophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-25) To a solution ofN-(3-aminophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (90 mg, 0.18 mmol) in DCM (5 mL) was added TEA (44 mg, 0.4 mmol)and acryloyl chloride (30 mg, 0.3 mmol) at 0° C. The reaction mixturewas diluted with water and saturated aqueous NaHCO₃ and extracted withDCM. The organic layer was washed with brine, dried over Na₂SO₄,concentrated under reduced pressure and the residue was purified bysilica gel chromatography to afford the compound as a white solid (36mg,25%). MS m/z 527[M+H]⁺. 1H NMR (400 MHz, DMSO) δ 11.54 (s, 1H), 10.37(s, 1H), 9.98 (s, 1H), 7.80 (d, J=9.0 Hz, 2H), 7.65 (d, J=9.0 Hz, 2H),7.30 (m, 4H), 6.90 (s, 1H), 6.63 (s, 1H), 6.48 (m, 1H), 6.20 (m, 2H),5.72 (d, J=10.0 Hz, 1H), 5.44 (q, J=16.0 Hz, 2H), 5.30 (d, J=16.5 Hz,1H), 4.68 (br, 1H), 4.35 (d, J=13.1 Hz, 1H), 3.37 (m, 1H), 3.10 (m, 1H),1.06 (d, J=6.9 Hz, 3H).

ISO-2-25-R and ISO-2-25-S were obtained by HPLC separation with a chiralcolumn.

The synthesis ofN-(1-acryloylpiperidin-4-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-26)

tert-butyl-4-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)piperidine-1-carboxylate(12) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.1 g, 0.26 mmol) in DMF (5 mL) were added tert-butyl4-aminopiperidine-1-carboxylate (32.6 mg, 0.28 mmol), HATU (0.15 g, 0.39mmol) and DIEA (0.067 g, 0.52 mmol). The mixture was stirred at roomtemperature under nitrogen. After 1 h, the reaction mixture was addedwater and the resulting precipitate was collected by filtration anddried to afford compound (12) (107 mg, 82%) as a white solid. MS m/z565[M+H]⁺.

1-(4-fluorobenzyl)-7-methyl-N-(piperidin-4-yl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a round-bottom flask containingtert-butyl-4-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)piperidine-1-carboxylate(12) (0.1 g, 0.18 mmol) was added with HCl (4 N in ethyl acetate, 5 mL).The resulting clear solution was stirred at room temperature. Thereaction mixture was concentrated under reduced pressure and theresulting residue was dried to afford the product (90 mg, 100%) as ayellow solid.

N-(1-acryloylpiperidin-4-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-26) To a solution of1-(4-fluorobenzyl)-7-methyl-N-(piperidin-4-yl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (90 mg, 0.18 mmol) in DCM (5 mL) was added with TEA (44 mg, 0.4mmol) and acryloyl chloride (30 mg, 0.3 mmol) at 0° C. The reactionmixture was diluted with water and saturated aqueous NaHCO₃ andextracted with DCM. The organic layer was washed with brine and dried.Then the mixture was then concentrated under reduced pressure and theresidue was purified by silica gel chromatography to afford the compoundas a white solid (35 mg, 37%). MS m/z 519[M+H]⁺. 1H NMR (400 MHz, DMSO)δ 11.52 (s, 1H), 8.01 (d, J=8.3 Hz, 1H), 7.20 (m, 4H), 6.90 (s, 1H),6.81 (m, 1H), 6.55 (m, 1H), 6.16 (m, 1H), 6.08 (d, J=16.7 Hz, 1H), 5.66(dd, J=10.5, 2.5 Hz, 1H), 5.40 (q, J=16.2 Hz, 2H), 5.24 (d, J=16.5 Hz,1H), 4.59 (br, 1H), 4.41 (m, 1H), 4.32 (d, J=11.3 Hz, 1H), 4.02 (m, 2H),3.30 (m, 1H), 3.11 (m, 2H), 2.69 (t, J=6.7 Hz, 1H), 1.76 (m, 2H), 1.50(m, 2H), 1.05 (d, J=6.8 Hz, 3H).

The synthesis of1-(3-acrylamidobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-27)

5-(tert-butyl)-3-ethyl-7-methyl-1-(3-nitrobenzyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) To a solution of 5-(tert-butyl)-3-ethyl7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(4) (1.24 g, 4 mmol) in dry THF (10 mL) was added NaH (60% wt dispersionin mineral oil, 0.2 g, 4.8 mmol) with stirring under nitrogen. After 1h, 1-(bromomethyl)-3-nitrobenzene (1.04 g, 4 mmol) was added and thereaction mixture was stirred 4 h under nitrogen. The reaction mixturewas quenched with water and the pH was adjusted to 6.0 with 1 N HCl. Themixture was diluted with brine and extracted with ethyl acetate. Thecombined extracts were dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The residue was purified by silica gelchromatography. The appropriate fractions were combined, concentratedunder reduced pressure and the residue was dried to afford the product.MS m/z 445[M+H]⁺.

Ethyl-7-methyl-1-(3-nitrobenzyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(7) To a round-bottom flask containing 5-(tert-butyl) 3-ethyl7-methyl-1-(3-nitrobenzyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) (1 g, 2.25 mmol) was added with HCl (4 N in ethyl acetate, 10 mL).The resulting clear solution was stirred at room temperature. Thereaction mixture was concentrated under reduced pressure and theresulting residue was dried to afford the product (0.85 g, 100%) as ayellow solid.

Ethyl-7-methyl-1-(3-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) To a solution of ethyl7-methyl-1-(3-nitrobenzyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(7) (0.85 g, 2.1 mmol) in DCM (10 mL) was added DIEA (0.67 g, 5.2 mmol)and 1H-pyrrole-2-carbonyl chloride (8A) (0.6 g, 2.5 mmol) at 0° C. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃ andextracted with DCM. The organic layer was washed with brine andconcentrated under reduced pressure and the residue was purified bysilica gel chromatography to afford the compound as a yellow solid (0.76g, 80%). MS m/z 438 [M+H]⁺.

7-methyl-1-(3-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) To a solution of ethyl7-methyl-1-(3-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) (0.1 g, 0.22 mmol) in MeOH (3 mL) was added with 5N LiOH (1 mL). Theclear solution was stirred at room temperature for lh. The reactionmixture was added with water and pH was adjusted to 6.0 with 1N HCl. Theresulting precipitate was collected by filtration and dried to affordthe product (70 mg, 74.5%) as a white solid. MS m/z 410 [M+H]⁺.

7-methyl-1-(3-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of7-methyl-1-(3-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (70 mg, 0.16 mmol) in DMF (2 mL) were added m-toluidine (25mg, 0.18 mmol), HATU (92 mg, 0.24 mmol) and DIEA (42 mg, 0.32 mmol). Themixture was stirred at room temperature under nitrogen. After 1 h, thereaction mixture was added with water and the resulting precipitate wascollected by filtration and dried to afford compound (12) (74 mg, 85%)as a white solid. MS m/z 499[M+H]⁺.

1-(3-aminobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of7-methyl-1-(3-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.4 g, 0.73 mmol) in ethyl acetate/methanol (4/1)(10mL) was addedstannous chloride (1.11 g, 5.86 mmol). The resulting mixture was heatedat 80° C. The reaction mixture was added DCM and the pH adjusted to 8with saturated aqueous NaHCO₃ and extracted with DCM. The organic layerwas washed with brine and dried. Then concentrated under reducedpressure and the residue was purified by silica gel chromatography toafford the compound as a white solid (0.36 g, 95.5%). MS m/z 469[M+H]⁺.

1-(3-acrylamidobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-27) To a solution of1-(3-aminobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.36 g, 0.7 mmol) in DCM (5 mL) was added DIEA (0.18 g, 1.4 mmol)and acryloyl chloride (95 mg, 1.05 mmol) at 0° C. The reaction mixturewas diluted with water and saturated aqueous NaHCO₃ and extracted withDCM. The organic layer was washed with brine and then concentrated underreduced pressure and the residue was purified by silica gelchromatography to afford the compound as a white solid (60 mg, 15%).MSm/z 523 [M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.54 (s, 1H), 10.40 (s, 1H),9.88 (s, 1H), 7.69 (m, 2H), 7.58 (d, J=8.2 Hz, 1H), 7.52 (s, 1H), 7.31(t, J=7.9 Hz, 1H), 7.18 (t, J=7.8 Hz, 1H), 6.88 (m, 3H), 6.60 (s, 1H),6.49 (m, 1H), 6.27-6.15 (m, 2H), 5.72 (d, J=10.1 Hz, 1H), 5.44 (q,J=16.2 Hz, 2H), 5.32 (d, J=16.6 Hz, 1H), 4.67 (br, 1H), 4.36 (d, J=11.6Hz, 1H), 3.40 (br, 1H), 3.18 (s, 1H), 2.29 (s, 3H), 1.11 (d, J=6.8 Hz,3H).

The synthesis of1-(4-acrylamido-3-methylbenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-28)

5-(tert-butyl)-3-ethyl-7-methyl-1-(3-methyl-4-nitrobenzyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) To a solution of 5-(tert-butyl) 3-ethyl7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(4) (1.5 g, 4.85 mmol) in dry THF (20 mL) was added NaH (60% wtdispersion in mineral oil, 0.23 g, 5.82 mmol) with stirring undernitrogen. After 1 h, 4-(bromomethyl)-2-methyl-1-nitrobenzene (1.26 g,4.85 mmol) was added and the reaction mixture was stirred for 4 h undernitrogen. The reaction mixture was quenched with water and the pH wasadjusted to 6.0 with 1 N HCl. The mixture was diluted with brine andextracted with ethyl acetate. The organic layer was dried andconcentrated. The residue was purified by silica gel chromatography toafford 5-(tert-butyl) 3-ethyl7-methyl-1-(3-methyl-4-nitrobenzyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(0.91 g, 40.6%) as a light yellow solid. MS m/z 459[M+H]⁺.

Ethyl-7-methyl-1-(3-methyl-4-nitrobenzyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(7) To a round-bottom flask containing 5-(tert-butyl) 3-ethyl7-methyl-1-(3-methyl-4-nitrobenzyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(6) (0.91 g, 2 mmol) was added with HCl (4 N in ethyl acetate, 5 mL).The resulting clear solution was stirred at room temperature. Thereaction mixture was concentrated under reduced pressure and theresulting residue was dried to afford ethyl7-methyl-1-(3-methyl-4-nitrobenzyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(0.78 g, 100%) as a yellow solid.

Ethyl-7-methyl-1-(3-methyl-4-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) To a solution of ethyl 7-methyl-1-(3-methyl-4-nitrobenzyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate (7) (0.8 g, 2.5mmol) in DCM (10 mL) was added DIEA (0.65 g, 6.25 mmol) and1H-pyrrole-2-carbonyl chloride (0.31 g, 3 mmol) at 0° C. for 10 min. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃ andextracted with DCM. The organic layer was washed with brine and dried.Then concentrated under reduced pressure and the residue was purified bysilica gel chromatography to afford the compound as a yellow solid (0.8g, 73%). MS m/z 452[M+H]⁺.

7-methyl-1-(3-methyl-4-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) To a solution of ethyl7-methyl-1-(3-methyl-4-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(9) (0.8 g, 1.76 mmol) in 1,4-dioxane (5 mL) was added with 5 N LiOH (5mL). The clear solution was stirred at room temperature. The reactionmixture was added with water and pH was adjusted to 6.0 with 1 N HCl.The resulting precipitate was collected by filtration and dried toafford7-methyl-1-(3-methyl-4-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (0.7 g, 92%) as a white solid. MS m/z 424[M+H]⁺.

7-methyl-1-(3-methyl-4-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of7-methyl-1-(3-methyl-4-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.32 g, 0.75 mmol) in DMF (5 mL) were added m-toluidine (0.1g, 0.9 mmol), HATU (0.43 g, 1.125 mmol) and DIEA (0.2 g, 1.5 mmol). Themixture was stirred at room temperature under nitrogen. After 1 h, thereaction mixture was added with water and the resulting precipitate wascollected by filtration and dried to afford compound (12) (0.36 g, 95%)as a white solid. MS m/z 513[M+H]⁺.

1-(4-amino-3-methylbenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of7-methyl-1-(3-methyl-4-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.36 g, 0.7 mmol) in ethyl acetate/methanol (4/1, 10 mL) was addedTin(II) chloride (1.12 g, 5.6 mmol). The resulting mixture was heated at80° C. overnight. The reaction mixture was added DCM and the pH wasadjusted to 8.0 with saturated aqueous NaHCO₃ and extracted with DCM.The organic layer was washed with brine and dried. Then concentratedunder reduced pressure and the residue was purified by silica gelchromatography to afford the compound as a white solid (0.3 g, 88%). MSm/z 483[M+H]⁺.

1-(4-acrylamido-3-methylbenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-28) To a solution of1-(4-amino-3-methylbenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.3 g, 0.62 mmol) in DCM (10 mL) was added TEA (0.19 g, 1.86 mmol)and acryloyl chloride (0.17 g, 1.86 mmol) at 0° C. The reaction mixturewas diluted with water and saturated aqueous NaHCO₃ and extracted withDCM. The organic layer was washed with brine and dried. Thenconcentrated under reduced pressure and the residue was purified bysilica gel chromatography to afford the compound as a white solid (86mg, 26%). MS m/z 537 [M+], H NMR (400 MHz, DMSO), δ 11.54 (s, 1H), 9.88(s, 1H), 9.53 (s, 1H), 7.69 (s, 1H), 7.56 (d, J=8.5 Hz, 1H), 7.46 (d,J=8.2 Hz, 1H), 7.19 (t, J=7.8 Hz, 1H), 7.10 (s, 1H), 6.99 (d, J=8.2 Hz,1H), 6.91 (m, 2H), 6.56 (m, 1H), 6.49 (m, 1H), 6.28-6.15 (m, 2H), 5.73(d, J=10.2 Hz, 1H), 5.40 (q, J=16.0 Hz, 2H), 5.31 (d, J=16.2 Hz, 1H),4.67 (br, 1H), 4.36 (d, J=11.0 Hz, 1H), 3.30 (m, 1H), 3.18 (s, 1H), 2.29(s, 3H), 2.19 (s, 3H), 1.12 (d, J=6.8 Hz, 3H).

The synthesis ofN-(3-acrylamido-4-chlorophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-29)

N-(4-chloro-3-nitrophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.1 g, 0.26 mmol) in DMF (5 mL) were added4-chloro-3-nitroaniline (32 mg, 0.28 mmol), HATU (0.15 g, 0.39 mmol) andDIEA (0.37 g, 0.52 mmol). The mixture was stirred at room temperatureunder nitrogen. After 1 h, the reaction mixture was added with water andthe resulting precipitate was collected by filtration and dried toafford compound (12) (115 mg, 82%) as a white solid. MS m/z 537[M+H]⁺.

N-(3-amino-4-chlorophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution ofN-(4-chloro-3-nitrophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.4 g, 0.7 mmol) in ethyl acetate/methanol (4/1)(10 mL) was addedstannous chloride (1.12 g, 5.6 mmol). The resulting mixture was heatedat 80° C. and dichloromethane was added , the pH adjusted to 8 andextracted with dichloromethane. The organic layer was washed with brine,dried over Na₂SO₄, concentrated under reduced pressure and the residuewas purified by silica gel chromatography to afford the compound as awhite solid (0.31 g, 82%). MS m/z507[M+H]⁺.

N-(3-acrylamido-4-chlorophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-29) To a solution ofN-(3-amino-4-chlorophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (110 mg, 0.21 mmol) in dichloromethane (5 mL) was added TEA (48 mg,0.42 mmol) and acryloyl chloride (35 mg, 0.33 mmol) at 0° C. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃.Dichloromethane was added and the organic layer was washed with brineand dried. Then concentrated under reduced pressure and the residue waspurified by silica gel chromatography to afford the compound as a whitesolid (28 mg, 20%). MS m/z 561[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.54 (s,1H), 10.24 (s, 1H), 9.79 (s, 1H), 8.29 (d, J=2.3 Hz, 1H), 7.69 (dd,J=8.8, 2.5 Hz, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.27 (m, 2H), 7.19 (m, 2H),6.91 (m, 1H), 6.69-6.55 (m, 2H), 6.28 (dd, J=17.2, 2.0 Hz, 1H), 6.17 (m,1H), 5.79 (dd, J=10.0, 1.6 Hz, 1H), 5.45 (q, J=16.0 Hz, 2H), 5.30 (d,J=16.4 Hz, 1H), 4.67 (br, 1H), 4.35 (d, J=12.0 Hz, 1H), 3.37 (m, 1H),3.17 (m, 1H), 1.08 (d, J=6.8 Hz, 3H).

The synthesis ofN-(3-acrylamido-4-methoxyphenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-30)

1-(4-fluorobenzyl)-N-(4-methoxy-3-nitrophenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of 1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.1 g, 0.26 mmol) in DMF (5mL) were added4-methoxy-3-nitroaniline (32 mg, 0.28 mmol), HATU (0.15 g, 0.39 mmol)and DIEA (0.37 g, 0.52 mmol). The mixture was stirred at roomtemperature under nitrogen. After 1 h, the reaction mixture was addedwith water and the resulting precipitate was collected by filtration anddried to afford compound (12) (105 mg, 78%) as a white solid. MS m/z533[M+H]⁺.

N-(3-amino-4-methoxyphenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of1-(4-fluorobenzyl)-N-(4-methoxy-3-nitrophenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.35 g, 0.66 mmol) in ethyl acetate/methanol (4/1)(10 mL) wasadded stannous chloride (1.12 g, 5.6 mmol). The resulting mixture washeated at 80° C. DCM was added and pH was adjusted to 8. The organiclayer was washed with brine, dried over Na₂SO₄, concentrated underreduced pressure and the residue was purified by silica gelchromatography to afford the compound as a white solid (0.23 g, 75%). MSm/z503[M+H]⁺.

N-(3-acrylamido-4-chlorophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-30) To a solution ofN-(3-amino-4-methoxyphenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (110 mg, 0.21 mmol) in dichloromethane (5 mL) was added TEA (48 mg,0.42 mmol) and acryloyl chloride (35 mg, 0.33 mmol) at 0° C. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃.Dichloromethane was added and the organic layer was washed with brineand dried. Then concentrated under reduced pressure and the residue waspurified by silica gel chromatography to afford the compound as a whitesolid (42 mg, 36%).MS m/z 557[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.53 (s,1H), 9.87 (s, 1H), 9.37 (s, 1H), 8.44 (s, 1H), 7.51 (dd, J=8.9, 2.5 Hz,1H), 7.27 (m, 2H), 7.20 (m, 2H), 7.00 (d, J=9.0 Hz, 1H), 6.91 (m, 1H),6.69 (m, 1H), 6.59 (m, 1H), 6.28 (dd, J=17.2, 2.0 Hz, 1H), 6.17 (m, 1H),5.79 (dd, J=10.0, 1.6 Hz, 1H), 5.45 (q, J=16.4 Hz, 2H), 5.30 (d, J=16.4Hz,1H), 4.67 (br, 1H), 4.34 (d, J=12.0 Hz, 1H), 3.83 (s, 3H), 3.39 (s,1H), 3.22-3.10 (m, 1H), 1.08 (d, J=6.8 Hz, 3H).

ISO-2-30-R and ISO-2-30-S were obtained by HPLC purification on a chiralcolumn.

The synthesis ofN-(1-acryloylindolin-6-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-31)

Tert-butyl 6-nitroindoline-1-carboxylate (b) To a solution of6-nitroindoline (a) (3 g, 18.3 mmol) in DCM (30mL) was addeddi-tert-butyl dicarbonate (4.8 g, 22 mmol) and TEA (3.7 g, 36.6 mmol).The solution was stirred for 5 h at room temperature then washed withwater and brine. The organic layer was dried, filtered and concentrated.Silica gel column chromatography provided tert-butyl6-nitroindoline-1-carboxylate (b) as a white solid (2.4 g, 50%).

Tert-butyl 6-aminoindoline-1-carboxylate(11) A mixture of tert-butyl6-nitroindoline-1-carboxylate (b) (2.4 g, 9.1 mmol) and 10% Pd-C (0.1 g)in MeOH (10 mL) is stirred under 1 atmosphere pressure of hydrogen for 2h. The mixture is filtered and concentrated. Silica gel columnchromatography provided tert-butyl 6-aminoindoline-1-carboxylate (11) asa solid (2 g, 94%). MS m/z235[M+H]⁺.

Tert-butyl6-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)indoline-1-carboxylate(12)To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.2 g, 0.52 mmol) in DMF (5 mL) were added tert-butyl6-aminoindoline-1-carboxylate (11) (0.13 g, 0.57 mmol), HATU (0.3 g,0.78 mmol) and DIEA (0.13 g, 1.04 mmol). The mixture was stirred at roomtemperature under nitrogen. After 1 h, the reaction mixture was addedwater and the resulting precipitate was collected by filtration anddried on a lyophilizer overnight to afford tert-butyl6-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)indoline-1-carboxylate(12)as a white solid. MS m/z599[M+H]⁺.

1-(4-fluorobenzyl)-N-(indolin-6-yl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a round-bottom flask containing tert-butyl6-(1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamido)indoline-1-carboxylate(12)(0.33 g, 0.55 mmol) was added HCl (4 N in ethyl acetate,10 mL). Theresulting clear solution was stirred at room temperature for 3 h. Thereaction mixture was concentrated under reduced pressure and theresulting residue was dried to constant weight under high vacuum toafford1-(4-fluorobenzyl)-N-(indolin-6-yl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.27 g, 100%) as a solid. MS m/z499[M+H]⁺.

N-(1-acryloylindolin-6-yl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-31)To a solution of1-(4-fluorobenzyl)-N-(indolin-6-yl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.27 g, 0.54 mmol) in DCM (5 mL) was added DIEA (140 mg, 1.08mmol) and acryloyl chloride (98 mg, 1.08 mmol) at −20° C. for 5 min. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃ andextracted with two portions of DCM. The organic layer was washed withbrine, dried and concentrated under reduced pressure and the residue waspurified by silica gel chromatography to afford the compound as a whitesolid. MS m/z553[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.52 (s, 1H), 9.90 (s,1H), 8.64 (s, 1H), 7.44 (d, J=7.7 Hz, 1H), 7.28 (m, 2H), 7.23-7.13 (m,3H), 6.91 (m, 1H), 6.75 (m, 1H), 6.60 (m, 1H), 6.31 (d, J=17.2, 2.0 Hz ,1H), 6.18 (m, 1H), 5.82 (dd, J=10.2, 1.6 Hz, 1H), 5.44 (q, J=16.4 Hz,2H), 5.30 (d, J=16.8 Hz, 1H), 4.68 (br, 1H), 4.35 (d, J=12.0 Hz, 1H),4.22 (s, 2H), 3.38 (m, 1H), 3.10 (m, 3H), 1.08 (d, J=6.8 Hz, 3H).

The synthesis ofN-(3-acrylamidobenzyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-32)

1-(azidomethyl)-3-nitrobenzene (b) A stock solution of 0.5 M NaN3 inDMSO was prepared after stirring for 24 h at room temperature. A 250 mLround-bottom flask equipped with a magnetic stir bar, was charged with a0.5 M solution of NaN₃ (2.26 g, 34.7 mmol) in DMSO (70 mL) followed by1-(bromomethyl)-3-nitrobenzene (5 g, 23.1 mmol). The mixture was stirredovernight at room temperature, treated with H₂O and stirred until it wascooled to room temperature. The mixture was extracted with ethyl acetate(3 times). The ethyl acetate extracts were combined, washed with H₂O andbrine, dried, filtered and evaporated under reduced pressure.

(3-nitrophenyl)methanamine (11) A stirred mixture of1-(azidomethyl)-3-nitrobenzene (4.15 g, 23.3 mmol), CeCl₃ (8.6 g, 34.9mmol), and Nal (31.4 g, 209.7 mmol) in acetonitrile (230 mL) was stirredfor lh at 100° C. The reaction mixture was concentrated under reducedpressure and the resulting residue was dried under high vacuum toprovide (3-nitrophenyl)methanamine (11).

1-(4-fluorobenzyl)-7-methyl-N-(3-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) To a solution of1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.2 g, 0.52 mmol) in DMF (5 mL) were added(3-nitrophenyl)methanamine (11) (87 mg, 0.57 mmol), HATU (0.3 g, 0.78mmol) and DIEA (0.13 g, 1.04 mmol). The mixture was stirred at roomtemperature under nitrogen. After 1 h, the reaction mixture was addedwater and the resulting precipitate was collected by filtration anddried on a lyophilizer overnight to afford1-(4-fluorobenzyl)-7-methyl-N-(3-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) as a white solid. MS m/z517[M+H]⁺.

N-(3-aminobenzyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) To a solution of1-(4-fluorobenzyl)-7-methyl-N-(3-nitrobenzyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.3 g, 0.58 mmol) in ethyl acetate/methanol (4/1)(10 mL) was addedSnCl₂ (0.88 g, 4.64 mmol). The resulting mixture was heated at 80° C.overnight. The reaction mixture was added DCM and the pH adjusted to ˜8with saturated aqueous NaHCO₃ and extracted with DCM. The organic layerwas washed with brine, dried, concentrated under reduced pressure andthe residue was purified by silica gel chromatography to afford thecompound as a white solid. MS m/z487[M+H]⁺.

N-(3-acrylamidobenzyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-32) To a solution ofN-(3-aminobenzyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.18 g, 0.37 mmol) in dichloromethane (5 mL) was added DIEA (96mg, 0.74 mmol) and acryloyl chloride (67 mg, 0.74 mmol) at −20° C. for 5min. The reaction mixture was diluted with water and saturated aqueousNaHCO₃ and extracted with dichloromethane (DCM). The organic layer waswashed with brine, dried, concentrated under reduced pressure and theresidue was purified by silica gel chromatography to affordN-(3-acrylamidobenzyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamideas a white solid. MS m/z541[M+H]⁺.¹H NMR (400 MHz, DMSO) δ 11.50 (s,1H), 10.10 (s, 1H), 8.62 (t, J=6.3 Hz, 1H), 7.62 (d, J=8.2 Hz, 1H), 7.52(s, 1H), 7.28-7.21 (m, 3H), 7.18 (m, 2H), 7.00 (d, J=7.8 Hz, 1H), 6.90(m, 1H), 6.56 (m, 1H), 6.43 (m, 1H), 6.25 (dd, J=17.2, 2.0 Hz, 1H), 6.15(m, 1H), 5.73 (m, 1H), 5.37 (q, J=16.4 Hz, 2H), 5.26 (d, J=16.4 Hz, 1H),4.62 (br, 1H), 4.38 (d, J=6.3 Hz, 2H), 4.33 (d, J=12.0 Hz, 1H), 3.37 (m,1H), 3.19-3.08 (m, 1H), 1.08 (d, J=6.8 Hz, 3H).

The synthesis of(E)-1-(3-(4-chlorobut-2-enamido)-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-36)

5-((1H-pyrrol-2-yl)methyl)-1-(4-fluoro-3-nitrobenzyl)-7-methyl-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12)To a solution of1-(4-fluoro-3-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (10) (0.32 g, 0.75 mmol) in DMF (5 mL) were added m-toluidine (0.1g, 0.9 mmol), HATU (0.43 g, 1.125 mmol) and DIEA (0.2 g, 1.5 mmol). Themixture was stirred at room temperature under nitrogen. After 1 h, thereaction mixture was added water and the resulting precipitate wascollected by filtration and dried on a lyophilizer overnight to affordcompound (12) (0.36 g, 95%) as a white solid. MS m/z517[M+H]⁺.

1-(3-amino-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13)To a solution of5-((1H-pyrrol-2-yl)methyl)-1-(4-fluoro-3-nitrobenzyl)-7-methyl-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (0.36 g, 0.7 mmol) in ethyl acetate/methanol (4/1) 10 mL) was addedstannous chloride (1.12 g, 5.6 mmol). The resulting mixture was heatedat 80° C. overnight. The reaction mixture was added DCM and the pH wasadjusted to 8 with saturated aqueous NaHCO₃ and extracted with DCM. Theorganic layer was washed with brine, dried, concentrated under reducedpressure and the residue was purified by silica gel chromatography toafford the compound as a white solid (0.3 g, 88%). MS m/z487[M+H]⁺.

1-(3-amino-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-36)To a solution of1-(3-acrylamido-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (0.12 g, 0.247 mmol) in DCM (2 mL) was added DIEA (64 mg, 0.494mmol) and (E)-4-chlorobut-2-enoyl chloride (41 mg, 0.296 mmol) at −20°C. for 5 min. The reaction mixture was diluted with water and saturatedaqueous NaHCO₃ and extracted with DCM. The organic layer was washed withbrine, dried, concentrated under reduced pressure and the residue waspurified by silica gel chromatography to afford the compound as a whitesolid. MS m/z590[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.52 (s, 1H), 9.89 (s,1H), 9.83 (s, 1H), 7.83 (d, J=6.8 Hz, 1H), 7.68 (s, 1H), 7.56 (d, J=8.2Hz, 1H), 7.27-7.15 (m, 2H), 6.96 (m, 1H), 6.91 (m, 2H), 6.59 (m, 1H),6.42 (d, J=7.0 Hz, 1H), 6.17 (m, 1H), 6.12 (m, 1H), 5.41 (q, J=16.4 Hz,2H), 5.30 (d, J=16.4 Hz, 1H), 4.67 (br, 1H), 4.36 (d, J=12.0 Hz, 1H),3.39 (d, J=5.0 Hz, 3H), 3.18 (m, 1H), 2.29 (s, 3H), 1.11 (d, J=6.8 Hz,3H).

The synthesis of1-((6-acrylamidonaphthalen-1-yl)methyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-38)

5-(tert-butoxycarbonyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (5) To a solution of 5-(tert-butyl) 3-ethyl7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(4) (5 g, 16.1 mmol) in MeOH (30 mL) was added with 5N LiOH (20 mL). Theclear solution was stirred at room temperature. The reaction mixture wasadded with water and pH was adjusted to 6 with 6N HCl. The resultingprecipitate was collected by filtration and dried to afford5-(tert-butoxycarbonyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (4.5 g, 99%) as a white solid. MS m/z 282 [M+H]⁺.

Tert-butyl7-methyl-3-(m-tolylcarbamoyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate(7) To a solution of5-(tert-butoxycarbonyl)-7-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylicacid (5) (2.4 g, 8.5 mmol) in DMF (10 mL) were added m-toluidine (1.2 g,10.2 mmol), HATU (4.8 g, 12.75mmol) and DIEA (2.5 g, 17 mmol). Themixture was stirred at room temperature. The reaction mixture was addedwith water and the resulting precipitate was collected by filtration anddried to afford compound (7) (2.3 g, 74%) as a white solid. MS m/z371[M+H]⁺.

5-(tert-butyl) 3-ethyl1-(4-fluorobenzyl)-7-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylate(9) To a solution of tert-butyl7-methyl-3-(m-tolylcarbamoyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate(7) (1 g, 2.7 mmol) in dry DMSO (30 mL) was added K2CO3 (0.6 g, 4 mmol)and tert-butyl (5-(chloromethyl)naphthalen-2-yl)carbamate (1.5 g, 4mmol) . The mixture was stirred at 80° C. After 1 h the reaction mixturewas quenched with water and extracted with ethyl acetate. The combinedextracts were dried, filtered and concentrated under reduced pressure.The residue was purified by silica gel chromatography. The appropriatefractions were combined concentrated under reduced pressure and theresidue was dried to afford tert-butyl1-((6-((tert-butoxycarbonyl)amino)naphthalen-1-yl)methyl)-7-methyl-3-(m-tolylcarbamoyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate(1.05 g, 60%) as a light yellow solid. MS m/z 626 [M+H]⁺.

1-((6-aminonaphthalen-1-yl)methyl)-7-methyl-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(10) To a round-bottom flask containing tert-butyl1-((6-((tert-butoxycarbonyl)amino)naphthalen-1-yl)methyl)-7-methyl-3-(m-tolylcarbamoyl)-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate(9) (1 g, 1.6 mmol) was added with HCl (4 N in ethyl acetate, 20 mL).The resulting solution was stirred at room temperature. The reactionmixture was concentrated under reduced pressure and the resultingresidue was dried to afford1-((6-aminonaphthalen-1-yl)methyl)-7-methyl-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(0.75 g, 100%) as a yellow solid.

Ethyl1-(4-fluoro-3-nitrobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxylate(12)To a solution of1-((6-aminonaphthalen-1-yl)methyl)-7-methyl-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(10) (0.75 g, 1.76mmol) in dichloromethane (10 mL) was added TEA (0.27g, 2.64 mmol) and 1H-pyrrole-2-carbonyl chloride (0.227 g, 1.76 mmol) at-20° C. for 50 min. The reaction mixture was diluted with water andextracted with dichloromethane. The organic layer was dried,concentrated under reduced pressure and the residue was purified bysilica gel chromatography to afford the compound as a yellow solid (0.4g, 44%). MS m/z 519[M+H]⁺.

1-((6-acrylamidonaphthalen-1-yl)methyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-38) To a solution of1-((6-aminonaphthalen-1-yl)methyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (110 mg, 0.21 mmol) in dichloromethane (5 mL) was added TEA (48 mg,0.42 mmol) and acryloyl chloride (35 mg, 0.33 mmol) at 0° C. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃.Dichloromethane was added and the organic layer was washed with brineand dried. Then concentrated under reduced pressure and the residue waspurified by silica gel chromatography to afford the compound as a whitesolid (25 mg, 20%). MS m/z 573 [M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.55(s, 1H), 10.82 (s, 1H), 9.91 (s, 1H), 8.48 (s, 1H), 8.23 (d, J=9.0 Hz,1H), 7.92 (d, J=8.4 Hz, 1H), 7.79 (d, J=8.2 Hz, 1H), 7.69 (s, 1H), 7.56(d, J=8.2 Hz, 1H), 7.40 (t, J=8.2 Hz, 1H), 7.20 (t, J=7.8 Hz, 1H),6.95-6.84 (m, 2H), 6.70-6.56 (m, 3H), 6.31 (dd, J=17.0, 1.6 Hz, 1H),6.17 (s, 1H), 6.05 (d, J=16.4 Hz, 1H), 5.95 (d, J=16.4 Hz, 1H), 5.80 (d,J=10.0 Hz, 1H), 5.35 (d, J=16.7 Hz, 1H), 4.75 (br, 1H), 4.34 (d, J=11.6Hz, 1H), 3.38 (m, 1H), 3.15 (m, 1H), 2.29 (s, 3H), 1.03 (d, J=6.8 Hz,3H).

The synthesis of(R)-1-((6-acrylamidonaphthalen-1-yl)methyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-38-R)

The 12R was purified by chiral column and then the final compoundISO-2-38-R was made with same procedure as ISO-2-38.

The synthesis of(S)-1-((6-acrylamidonaphthalen-1-yl)methyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-38-S)

The 12S was purified by chiral column and then the final compoundISO-2-38-S was made with same procedure as ISO-2-38.

The synthesis of(E)-1-((6-(4-(dimethylamino)but-2-enamido)naphthalen-1-yl)methyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-39)

(E)-1-((6-(4-(dimethylamino)but-2-enamido)naphthalen-1-yl)methyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-39) To a solution of1-((6-aminonaphthalen-1-yl)methyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide (12) (200mg, 0.38 mmol) in dichloromethane (5 mL) was added DIEA (100 mg, 0.78mmol) and (E)-4-bromobut-2-enoyl chloride (106 mg, 0.78 mmol) at 0° C.10 min later dimethylamine (2 N in THF, 10 mL) was added. The reactionmixture was diluted with water and saturated aqueous NaHCO₃,dichloromethane was added and the organic layer was washed with brineand dried. Then concentrated under reduced pressure and the residue waspurified by silica gel chromatography to afford the compound as a whitesolid (25 mg, 10%). MS m/z630 [M+H]+. ¹H NMR (400 MHz, DMSO-d6) δ 11.54(s, 1H), 10.53 (s, 1H), 9.90 (s, 1H), 8.43 (s, 1H), 8.23 (d, J=9.1 Hz,1H), 7.84 (m, 2H), 7.68 (s, 1H), 7.56 (d, J=8.2 Hz, 1H), 7.40 (t, J=8.2Hz, 1H), 7.20 (t, J=7.8 Hz, 1H), 6.99-6.87 (m, 2H), 6.87-6.77 (m, 1H),6.67 (d, J=7.1 Hz, 1H), 6.59 (m, 1H), 6.45 (d, J=15.6 Hz, 1H), 6.19 (s,1H), 6.05 (d, J=16.4 Hz, 1H), 5.87 (d, J=16.0 Hz, 1H), 5.34 (d, J=16.8Hz, 1H), 4.71 (br, 1H), 4.33 (d, J=13.6 Hz, 1H), 3.49 (s, 2H), 3.33 (m1H), 3.14 (m, 1H), 2.47 (s, 6H), 2.29 (s, 3H), 1.02 (d, J=6.8 Hz, 3H).

The synthesis of1-(4-fluoro-3-propionamidobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-1Rev )

1-(4-fluoro-3-propionamidobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-1Rev) To a solution of1-(3-acrylamido-4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(0.2 g, 0.41mmol) in DCM (10 mL) was added TEA (0.15 g, 1.40 mmol) andpropionyl chloride (0.13 g, 1.4 mmol) at 0° C. The reaction mixture wasdiluted with water and saturated aqueous NaHCO₃ and extracted with DCM.The organic layer was washed with brine, dried over Na₂SO₄, concentratedunder reduced pressure and the residue was purified by silica gelchromatography to afford the compound as a white solid (66 mg, 26%). MSm/z543[M+H]+. ¹H NMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 9.85 (s, 1H),9.78 (s, 1H), 7.84 (d, J=6.4 Hz, 1H), 7.68 (s, 1H), 7.58 (d, J=8.0 Hz,1H), 7.28-7.11 (m, 2H), 6.98-6.81 (m, 3H), 6.58 (s, 1H), 6.16 (s, 1H),5.48-5.23 (m, 3H), 4.64 (br, 1H), 4.35 (d, J=12.4 Hz, 1H), 3.41 (m, 1H),3.28 (m, 1H), 2.38 (q, J=7.6 Hz, 2H), 2.28 (s, 3H), 1.10 (d, J=6.8 Hz,3H), 1.05 (t, J=7.5 Hz, 3H).

The synthesis of1-(4-fluorobenzyl)-7-methyl-N-(3-propionamidophenyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-4Rev)

1-(4-fluorobenzyl)-7-methyl-N-(3-propionamidophenyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-4Rev) To a solution ofN-(3-aminophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (180 mg, 0.37 mmol) in DCM (20 mL) was added TEA (75 mg, 0.6 mmol)and propionyl chloride (50 mg, 0.4 mmol) at 0° C. The reaction mixturewas diluted with water and saturated aqueous NaHCO₃ and extracted withDCM. The organic layer was washed with brine, dried over Na₂SO₄,concentrated under reduced pressure and the residue was purified bysilica gel chromatography to afford the compound as a white solid (42mg, 35%). MS m/z529[M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 11.6 (s, 1H),9.93 (s, 1H), 9.90 (s, 1H), 8.15 (s, 1H), 7.43 (d, J=8.0, Hz, 1H),7.37-7.24 (m, 3H), 7.19 (m, 3H), 6.91 (s, 1H), 6.58 (m, 1H), 6.18 (m,1H), 5.45 (q, J=16.4 Hz, 2H), 5.31 (d, J=16.4 Hz, 1H), 4.67 (br, 1H),4.35 (m, 1H), 3.38 (d, J=13.2 Hz, 1H), 3.21-3.10 (m, 1H), 2.32 (m, 2H),1.12-1.00 (m, 6H).

The synthesis of1-(4-fluorobenzyl)-7-methyl-N-(4-propionamidophenyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-25Rev)

1-(4-fluorobenzyl)-7-methyl-N-(4-propionamidophenyl)-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-25Rev) To a solution ofN-(3-aminophenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (90 mg, 0.18 mmol) in DCM (5 mL) was added TEA (44 mg, 0.4 mmol)and propionyl chloride (30 mg, 0.3 mmol) at 0° C. The reaction mixturewas diluted with water and saturated aqueous NaHCO₃ and extracted withDCM. The organic layer was washed with brine and dried. Thenconcentrated under reduced pressure and the residue was purified bysilica gel chromatography to afford the compound as a white solid (36mg, 25%). MS m/z529[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.53 (s, 1H), 9.94(s, 1H), 9.86 (s, 1H), 7.70 (d, J=9.0 Hz, 2H), 7.53 (d, J=8.9 Hz, 2H),7.26 (m, 2H), 7.19 (m, 2H), 6.91 (s, 1H), 6.58 (s, 1H), 6.16 (m, 1H),5.44 (q, J=16.4 Hz, 2H), 5.30 (d, J=16.4 Hz, 1H), 4.65 (br, 1H), 4.35(d, J=12.0 Hz, 1H), 3.38 (m, 1H), 3.17 (m, J=5.0 Hz, 1H), 2.36-2.24 (m,2H), 1.12-1.04 (m, 6H).

The synthesis of1-(4-fluorobenzyl)-N-(4-methoxy-3-propionamidophenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-30Rev)

1-(4-fluorobenzyl)-N-(4-methoxy-3-propionamidophenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-30Rev) To a solution ofN-(3-amino-4-methoxyphenyl)-1-(4-fluorobenzyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(13) (110 mg, 0.21 mmol) in dichloromethane (5 mL) was added TEA (48 mg,0.42 mmol) and propionyl chloride (35 mg, 0.33 mmol) at 0° C. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃.Dichloromethane was added and the organic layer was washed with brineand dried. Then concentrated under reduced pressure and the residue waspurified by silica gel chromatography to afford the compound as a whitesolid (42 mg, 36%). MS m/z559[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.53 (s,1H), 9.82 (s, 1H), 9.01 (s, 1H), 8.34 (s, 1H), 7.47 (m, 1H), 7.33-7.23(m, 2H), 7.23-7.14 (m, 2H), 6.97 (d, J=9.0 Hz, 1H), 6.91 (m, 1H), 6.58(m, 1H), 6.17 (m, 1H), 5.43 (q, J=16.4 Hz, 2H), 5.29 (d, J=16.4 Hz, 1H),4.66 (s, 1H), 4.34 (m, 1H), 3.81 (s, 3H), 3.37 (m, 1H), 3.16 (m, 1H),2.39 (m, 2H), 1.07 (m, 6H).

The synthesis of7-methyl-1-((6-propionamidonaphthalen-1-yl)methyl)-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-38Rev)

7-methyl-1-((6-propionamidonaphthalen-1-yl)methyl)-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(ISO-2-38Rev) To a solution of1-((6-aminonaphthalen-1-yl)methyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-N-(m-tolyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3-carboxamide(12) (110 mg, 0.21 mmol) in dichloromethane (5 mL) was added TEA (48 mg,0.42 mmol) and propionyl chloride (35 mg, 0.33 mmol) at 0° C. Thereaction mixture was diluted with water and saturated aqueous NaHCO₃.Dichloromethane was added and the organic layer was washed with brineand dried. Then concentrated under reduced pressure and the residue waspurified by silica gel chromatography to afford the compound as a yellowsolid (25 mg, 20%). MS m/z575[M+H]⁺. ¹H NMR (400 MHz, DMSO) δ 11.53 (s,1H), 10.20 (s, 1H), 9.88 (s, 1H), 8.35 (s, 1H), 8.19 (d, J=9.1 Hz, 1H),7.75 (m, 2H), 7.68 (s, 1H), 7.56 (d, J=7.4 Hz, 1H), 7.38 (t, J=8.0 Hz,1H), 7.18 (t, J=7.8 Hz, 1H), 6.91 (m, 2H), 6.64 (d, J=7.2 Hz, 1H), 6.59(s, 1H), 6.17 (m, 1H), 6.05 (d, J=16.4 Hz, 1H), 5.85 (d, J=16.4 Hz, 1H),5.34 (d, J=16.4 Hz, m, 1H), 4.71 (br, 1H), 4.33 (d, J=12.0 Hz, 1H), 3.38(m, 1H), 3.13 (m, 1H), 2.40 (q, J=7.2 Hz, 2H), 2.29 (s, 3H), 1.13 (t,J=7.6 Hz, 3H), 1.02 (d, J=6.8 Hz, 3H).

Separation of Enantiomers

Column CHIRALPAK IE(IE00CD-TB005) Column size 0.46 cm I.D. × 15 cm LInjection 10.0 μL or 20 μL Mobile phase DCM/MeOH = 95/5(V/V) Flow rate1.0 mL/min Wave length UV 254 nm Temperature 35° C. HPLC equipmentShimadzu LC-20AD CP-HPLC-07 HPLC equipment Shimadzu LC-20AD CP-HPLC-06

MALDI TOF Method for IsoCure Intact Mw Analysis

Experimental: Analyses were performed on a Shimadzu Biotech Axima TOF²(Shimadzu Instruments) matrix-assisted-laser desorption/ionizationtime-of-flight (MALDI-TOF) mass spectrometer. Proteins were analyzed inpositive ion linear mode. For intact protein mass measurement theinstrument was set with a mass range extending up to 75000 m/z using apulsed extraction setting of 47000 and apomyoglobin as the standard tocalibrate the instrument. A 3 ul aliquot of each sample was diluted with7 μL of 0.1% TFA prior to micro C4 Zip Tip desalting and depositiondirectly onto the MALDI target using Sinapinic acid as the desorptionmatrix (10 mg/mL in 0.1%TFA:Acetonitrile 50:50).

In Solution Digestion

A 5 μL aliquot of each sample was added to 20 μL of 0.1% ProteaseMAXSurfactant (Promega) in 50 mM Ammonium Bicarbonate to initially dissolveand denature the proteins. An additional aliquot of 20 μL of 50 mMAmmonium Bicarbonate was then added. For reduction a 2 μL aliquot of 45mM DTT was added and the samples were incubated at 50° C. for 30 min.For alkylation of cysteine a 2 μL aliquot of 100 mM iodoacetamide wasadded and the samples were incubated at room temperature for 30 min.Tryptic digestion was initiated with a 1 μL aliquot of Trypsin (0.2μg/μL Sigma Proteomics grade). Samples were incubated at 37° C. for 18h. Tryptic digests were acidified with 4 μL of 5% trifluoroacetic acid(TFA).

LC/MS/MS on Q Exactive

A 2.5 μL aliquot was directly injected onto a custom packed 2 cm×100 μmC₁₈ Magic 5μ particle trap column. Peptides were then eluted and sprayedfrom a custom packed emitter (75 μm×25 cm C₁₈ Magic 3 μm particle) witha linear gradient from 95% solvent A (0.1% formic acid in water) to 35%solvent B (0.1% formic acid in Acetonitrile) in 35 min at a flow rate of300 nanoliters per minute on a Waters Nano Acquity UPLC system. Datadependent acquisitions were performed on a Q Exactive mass spectrometer(Thermo Scientific) according to an experiment where full MS scans from300-1750 m/z were acquired at a resolution of 70,000 followed by 10MS/MS scans acquired under HCD fragmentation at a resolution of 17,500with an isolation width of 1.6 Da. Raw data files were processed withProteome Discoverer (version 2.1) prior to searching with Mascot Server(version 2.5) against the SwissProt Human database. Search parametersutilized were tryptic with 2 missed cleavages, parent mass tolerances of10 ppm and fragment mass tolerances of 0.05 Da. A fixed modification ofcarbamidomethyl cysteine and variable modifications of acetyl (proteinN-term), pyro glutamic for N-term glutamine, oxidation of methionine,and 3 different Isocure compounds were considered. Search results wereloaded into the Scaffold Viewer (Proteome Software, Inc.) for assessmentof protein identification probabilities and label free quantitation.

Testing for Biological Activities

Compounds were tested against 3 enzymes, as provided below.

Compounds were tested in 10-dose IC50 mode with 3-fold serial dilutionat a starting concentration of 10 μM.

Control Compounds were tested in 10-dose IC50 mode with 3-fold serialdilution starting at 1 mM or 10 mM.

Assay Format: The production or depletion of NADPH by IDH enzymes wasmeasured by diaphorase/resazurin coupled detection.

Fluorescence measurement: Ex/Em=535/590 by EnVision.

Compounds were pre-incubated with enzyme for 1 h at room temperature.

Standard substrate: 1500 μM a-Ketoglutarate+15 μM NADPH for IDH1(R132H). 500 μM a-Ketoglutarate+15 μM NADPH for IDH1 (R132C). 65 μMIsocitrate+50 μM NADP for IDH1.

Exemplary results are presented in Table 1 and FIGS. 2-7.

TABLE 1 Summary of Exemplary IC50 Results Compound IC50 (nM) CompoundID: IDH1 (R132H) IDH1 (R132C) IDH1 ISO-2-1 18 35 >10000 ISO-2-2 33152 >10000 ISO-2-3 34 77 10000 ISO-2-4 49 121 >10000 ISO-2-5 3 2 >10000ISO-2-6 >10000 >10000 >10000 ISO-2-7 153 247 >10000 ISO-2-8 52154 >10000 ISO-2-9 >10000 >10000 >10000 ISO-2-10 >10000 9500 >10000ISO-2-19 5.0 35 4780 ISO-2-20 5480 7370 >10000 ISO-2-25 0.6 0.8 5740ISO-2-25-R 10 21 2140 ISO-2-25-S 1360 7720 ISO-2-26 >10000 >10000 >10000ISO-2-27 25 127 >10000 ISO-2-28 149 5550 >10000 ISO-2-29 14 61 ISO-2-301 8 ISO-2-30-R 6 14 2210 ISO-2-30-S 839 1180 ISO-2-31 5 27 ISO-2-32 31507500 ISO-2-36 0.5 8 ISO-2-38 0.5 4

Applicant's disclosure is described herein in preferred embodiments withreference to the Figures, in which like numbers represent the same orsimilar elements. Reference throughout this specification to “oneembodiment,” “an embodiment,” or similar language means that aparticular feature, structure, or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent invention. Thus, appearances of the phrases “in one embodiment,”“in an embodiment,” and similar language throughout this specificationmay, but do not necessarily, all refer to the same embodiment.

The described features, structures, or characteristics of Applicant'sdisclosure may be combined in any suitable manner in one or moreembodiments. In the description, herein, numerous specific details arerecited to provide a thorough understanding of embodiments of theinvention. One skilled in the relevant art will recognize, however, thatApplicant's composition and/or method may be practiced without one ormore of the specific details, or with other methods, components,materials, and so forth. In other instances, well-known structures,materials, or operations are not shown or described in detail to avoidobscuring aspects of the disclosure.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

Equivalents

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

1. A compound having the structural formula of (I):

wherein, each of Ring A, Ring B and Ring C is independently a 5- or 6-membered cyclic or bicyclic alkyl, heteroalkyl, aryl, or heteroaryl ring; each R_(W) is independently a hydrogen, a halide, OH, NH₂, alkyl, alkyl amine, alkyl amide, alkoxy, or an electrophilic group R_(E); R₁ is H, halide, alkyl amine, methoxy, hydroxy or a C₁-C₃ alkyl group; X is CH₂, NH, O, S or a single bond; Y is CH₂, NH, O or S; and Z is CH₂, NH, O or S, or a pharmaceutically acceptable form thereof.
 2. The compound of claim 1, having the structure of:


3. The compound of claim 1, wherein R_(W) is R_(E).
 4. The compound of claim 1, having the structural formula of (I-A)


5. The compound of claim 2, having the structural formula of (II-A)


6. The compound of claim 1, having the structural formula of (I-B).


7. The compound of claim 2, having the structural formula of (I-B).


8. The compound of claim 1, having the structural formula of (I-C):


9. The compound of claim 2, having the structural formula of (I-C):


10. The compound of claim 1, wherein each of Ring A and Ring B is a 6-membered aryl group and Ring C is a 5-membered aryl group.
 11. The compound of claim 1, having the structural formula of:


12. The compound of claim 2, having the structural formula of:

13-36. (canceled)
 37. The compound of claim 1, wherein R_(E) comprises an acrylamide group.
 38. The compound of claim 1, wherein R_(E) comprises


39. The compound of claim 1, wherein R_(E) comprises


40. The compound of claim 1, wherein R_(E) comprises


41. The compound of claim 1, wherein R_(E) comprises

42-44. (canceled)
 45. A pharmaceutical composition comprising a compound having the structural formula of (I):

wherein, each of Ring A, Ring B and Ring C is independently a 5- or 6-membered cyclic or bicyclic alkyl, heteroalkyl, aryl, or heteroaryl ring; each R_(W) is independently a hydrogen, a halide, OH, NH₂, alkyl, alkyl amine, alkyl amide, alkoxy, or an electrophilic group R_(E); R₁ is H, halide, alkyl amine, methoxy, hydroxy or a C₁-C₃ alkyl group; X is CH₂, NH, O, S or a single bond; Y is CH₂, NH, O or S; and Z is CH₂, NH, O or S, or a pharmaceutically acceptable form thereof, effective to treat, prevent, or reduce one or more cancers, or a related disease or disorder thereof, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent. 46-47. (canceled)
 48. A unit dosage form comprising a pharmaceutical composition according to claim
 45. 49. A method for treating, reducing, or preventing a disease or disorder, comprising administering to a subject in need thereof a pharmaceutical composition comprising a compound having the structural formula of (I):

wherein, each of Ring A, Ring B and Ring C is independently a 5- or 6-membered cyclic or bicyclic alkyl, heteroalkyl, aryl, or heteroaryl ring; each R_(W) is independently a hydrogen, a halide, OH, NH₂, alkyl, alkyl amine, alkyl amide, alkoxy, or an electrophilic group R_(E); R₁ is H, halide, alkyl amine, methoxy, hydroxy or a C₁-C₃ alkyl group; X is CH₂, NH, O, S or a single bond; Y is CH₂, NH, O or S; and Z is CH₂, NH, O or S, or a pharmaceutically acceptable form thereof, effective to treat, prevent, or reduce one or more cancers, or a related disease or disorder thereof, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent. 50-51. (canceled) 